VK Malhotra - Biochemistry for Students, 12th Edition

BIOCHEMISTRY for Students

BIOCHEMISTRY for Students 12th Edition

VK Malhotra PhD (Gold Medalist) Department of Biochemistry Maulana Azad Medical College (MAMC) New Delhi, India

Foreword Nancy Kaul

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Biochemistry for Students © 2012, Jaypee Brothers Medical Publishers All rights reserved. No part of this publication should be reproduced, stored in a retrieval system, or transmitted in any form or by any means: electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the author and the publisher. This book has been published in good faith that the material provided by the author is original. Every effort is made to ensure accuracy of material, but the publisher, printer and author will not be held responsible for any inadvertent error (s). In case of any dispute, all legal matters are to be settled under Delhi jurisdiction only.

Previous Editions:

1978, 1980, 1982, 1984, 1985, 1987, 1989, 1991 (Reprint 1993), 1996, 1998, 2003 (Reprint 2006, 2008)

Twelfth Edition: 2012 ISBN 978-93-5025-504-9

Typeset at JPBMP typesetting unit Printed at

Foreword Biochemistry has been playing a very important role in dayto-day life of medical students. The book Biochemistry for Students written by Dr VK Malhotra, Gold Medalist, serves as a quick reading material being purposefully written in clear, lucid and precise manner. This book will certainly serve the needs of medical students. Dr (Mrs) Nancy Kaul Ex-Head, Department of Biochemistry Lady Hardinge Medical College New Delhi, India

Preface to the Twelfth Edition This book is revised keeping in view all categories of students and it addresses their needs in a simple and practical manner as biochemistry tries to explain the mystery of life in the language of chemistry. I hope the book will be received warmly by the students as well as teachers for both desire maximum benefits out of it. All the chapters are revised to gain understanding and clarity. Suggestions to improve the future editions are most welcome and will be highly appreciated. I would like to thank Shri Jitendar P Vij (Chairman and Managing Director) and Mr Tarun Duneja (Director Publishing) of M/s Jaypee Brothers Medical Publishers (P) Ltd, New Delhi for the publication of this book. Mr Subrata Adhikary (Author Coordinator) deserves special praise for this venture. VK Malhotra

Preface to the First Edition Biochemistry currently occupies an eminent position particularly among medical subjects. However, there are few texts in the market at present which enable the students to acquire a working knowledge of the subject. Having been connected with the teaching profession for the past few years, I am well acquainted with the difficulties encountered by the students while trying to master the subject. The present book is the result of my humble attempt to overcome these handicaps and present the subject in a simple and easily comprehensible form. Attempts have been made to illustrate the subject matter with diagrams and chemical formulae wherever necessary. Special thanks to my publisher Shri Jitendar P Vij, without whose help, this book could not have seen the light of the day. VK Malhotra

Contents 1.

Biophysics ......................................................................... 1 • • • • • •

2.

Chemistry of Carbohydrates ..................................... 19 • • • • • •

3.

Hydrogen Ion Concentration, pH ................................................. 1 Osmosis and Osmotic Pressure .................................................... 12 Colloids ............................................................................................ 16 Surface Tension ............................................................................... 17 Absorption ....................................................................................... 18 Viscosity ........................................................................................... 18

Carbohydrates ................................................................................ 19 Functions of Carbohydrates ......................................................... 19 Classification of Carbohydrates ................................................... 19 Oligosaccharides ............................................................................. 40 Polysaccharides ............................................................................... 45 Heteropolysaccharides .................................................................. 49

Chemistry of Lipids .................................................... 53 • Simple Lipids ................................................................................... 54 • Compound Lipids ........................................................................... 62 • Derived Lipid ................................................................................... 68

4.

Chemistry of Amino Acids and Proteins ............ 74 • Chemistry of Amino Acids ........................................................... 74 • Proteins ............................................................................................ 85

5.

Hemoglobin .................................................................. 102 • Porphins ......................................................................................... 102 • Porphyrins ..................................................................................... 103

xii BIOCHEMISTRY FOR STUDENTS • Hemoglobin .................................................................................. 103 • Porphyria ....................................................................................... 114

6.

Enzymes ........................................................................ 120 • • • • • • •

7.

Biological Oxidation ................................................. 140 • • • •

8.

Biological Oxidation ...................................................................... 140 Mixed Function Oxidases ............................................................. 142 High Energy Compounds ........................................................... 143 Respiratory Chain ........................................................................ 144

Metabolism of Carbohydrates ............................... 151 • • • • • • • • • •

9.

Enzymes ......................................................................................... 120 Factors Influencing the Rate of Enzymatic Reactions ............. 124 Enzyme Activity ........................................................................... 129 Enzyme Inhibitions ...................................................................... 130 Catalytic Site or the Active Sites of the Enzymes .................... 134 Enzyme Induction ........................................................................ 135 Diagnostic Value of Plasma Enzymes ....................................... 137

Glycolysis ....................................................................................... 151 Citric Acid Cycle ........................................................................... 155 Energetics ....................................................................................... 158 Glycogenesis .................................................................................. 163 Gluconeogenesis ........................................................................... 168 Galactose Metabolism .................................................................. 169 Fructose Metabolism .................................................................... 172 Lactose Synthesis .......................................................................... 173 Uronic Acid Pathway ................................................................... 174 Regulation of Blood Glucose ....................................................... 175

Metabolism of Lipids ............................................... 184 • Plasma Lipoproteins ..................................................................... 184

CONTENTS xiii

10. Metabolism of Proteins ........................................... 210 • • • • • • • •

Digestion and Absorption ........................................................... 210 Urea Cycle (Krebs-Henseleit Cycle) .......................................... 214 Glycine ............................................................................................ 221 Methionine ..................................................................................... 226 Cysteine and Cystine ................................................................... 227 Phenylalanine and Tyrosine ........................................................ 229 Tryptophan .................................................................................... 237 Leucine, Isoleucine and Valine .................................................... 240

11. Nucleic Acid—Chemistry and Metabolism ...... 241 • Nucleic Acids ................................................................................. 244

12. Vitamins ....................................................................... 259 • • • • • • • • • • • • • • • •

Fat Soluble Vitamins .................................................................... 261 Vitamin A ....................................................................................... 261 Vitamin D ....................................................................................... 264 Vitamin E ....................................................................................... 265 Vitamin K ....................................................................................... 266 Water Soluble Vitamins ............................................................... 268 Vitamin C ....................................................................................... 268 Thiamine ........................................................................................ 270 Riboflavin ....................................................................................... 272 Niacin .............................................................................................. 273 Pantothenic Acid ........................................................................... 275 Pyridoxine ...................................................................................... 275 Biotin ............................................................................................... 277 Folic Acid ........................................................................................ 279 Cyanocobalamin ........................................................................... 281 Antivitamins .................................................................................. 283

13. Acid-base Balance ..................................................... 284 • Acid-base Balance ......................................................................... 284

xiv BIOCHEMISTRY FOR STUDENTS

14. Water and Mineral Metabolism ........................... 292 • Biological Importance of Water .................................................. 292 • Minerals .......................................................................................... 295

15. Xenobiotics .................................................................... 306 16. Nutrition ....................................................................... 310 • Food Values ................................................................................... 319 • 1500 Calories Diabetic Diet Chart .............................................. 322

17. Organ Function Tests ............................................... 326 • • • •

Liver Function Tests ..................................................................... 326 Renal Function Tests .................................................................... 330 Pancreatic Function Test .............................................................. 335 Gastrointestinal (Git) Function Test ........................................... 338

18. Immunology ................................................................. 339 • Introduction ................................................................................... 339 • Functions of T Cells ...................................................................... 343

19. Cancer ............................................................................ 356 20. Hormones...................................................................... 360 • • • • • •

Insulin ............................................................................................. 364 Glucagon ........................................................................................ 367 Triiodothyronine (T3) and Thyroxine (T4) ................................. 367 Calcitonin ....................................................................................... 368 Parathormone ............................................................................... 369 Thyroid Gland ............................................................................... 370

CONTENTS xv

21. Protein Biosynthesis ................................................. 371 • • • • • •

Activation Step .............................................................................. 372 Initiation of Polypeptide Chain (In Ribosomes) ...................... 374 Elongation ...................................................................................... 376 Termination ................................................................................... 378 Codon ............................................................................................. 380 Regulation of Gene Expression .................................................. 381

22. Instrumentation .......................................................... 385 • • • • • •

Colorimetry ................................................................................... 385 Electrophoresis .............................................................................. 386 Isotopes and their Application .................................................... 387 Electrometric Determination of pH ........................................... 388 Estimation of Nitrogen Content by Micro-Kjeldahl Method ..... 390 Chromatography ......................................................................... 393

Index ........................................................................................ 397

CHAPTER

Biophysics

1

HYDROGEN ION CONCENTRATION, pH Acids are substances which furnish hydrogen ions (H+) in the solution, whereas bases are substances that furnish hydroxide ions (OH–) in the solution. Substances that dissociate in water into a cation (positively charged ion) and an anion (negatively charged ion) are classified as electrolytes. Whereas sugar or alcohols which dissolve in water but do not carry a charge or dissociate into species with a positive and negative charge are classified as nonelectrolytes. Strong electrolytes are completely ionized in aqueous solutions whereas weak electrolytes are partially ionized in aqueous solutions. pH of a solution is defined as the negative logarithm of its hydrogen ion concentration.

pH = – log10 [H+] =

1 log 10 [H + ]

Pure water has equal concentration of H+ and OH– ions, the concentrations of each is very small and each being equal to 10–7 moles/liter at room temperature. Water dissociates into: H2O

H+ + OH–

From the Law of Mass action, the dissociation of water can be represented as:

Kw =

[H+ ] [OH – ] [H2 O]

2 BIOCHEMISTRY FOR STUDENTS

The bracket indicates the concentration of each component in moles per liter. The concentration of undissociated water is so large as compared to the concentration of H+ and OH– ions, so that for all the practical purposes it is fairly constant. This simplifies the above equation to: [H+] [OH–] = K [H2O] [H+] [OH–] = Kw Where Kw is ionic product of water or the dissociation constant of water. Electrical conductivity measurements have shown that dissociation constant of water is constant at a given temperature and changes with the change in temperature. Ionic product of water is usually taken as 10–14 at the room temperature (25ºC). Then [H+] [OH–] = 10–14 Taking logarithm of both sides log [H+] + log [OH–] = –14 By rearrangement –log [H+] –log [OH–] = 14 According to the definition of pH, the above equation simplifies to: pH + pOH = 14 At neutrality, both hydrogen and hydroxide ions have equal concentration, i.e. pH = 7 pOH = 7 There exists an inverse relationship between [H+] and [OH–] ions in solution. As hydrogen ion concentration increases, the hydroxide ion concentration decreases and vice versa. The acidity or alkalinity of a solution is determined by the amount of [H+] and [OH–] ions present.

BIOPHYSICS

3

A solution having hydrogen ions concentration of one normality (1 N) will have a pH 0, and other having hydroxide concentration of one normality (1 N) will have pH 14. It should also be kept in mind that a change of one pH unit brings a ten-fold change in acidity or alkalinity, i.e. a solution of pH 5 has ten times more the hydrogen ion concentration than that of a solution of pH 6 and a hundred times more than that of a solution of pH 7. If hydrogen ion concentration is doubled, the pH falls by 0.3 units. The average pH values of some of body fluids are: Gastric juice 1.4 Saliva 6.8 Urine 6.0 Milk 7.1 Tears 7.2 Blood 7.4 Pancreatic juice 8.0 Q. Calculate the pH of a solution of which hydrogen ion concentration is 4.6 × 10–9 M. Ans. pH = –log10 [CH+] = –log10 [4.6 × 10–9] = –log10 4.6 + 9 log10 10 = –0.66 + 9 = 8.34. Q. Calculate the hydrogen ion concentration of a solution, the pH of which is 4.50. Ans. pH = –log10 [CH+]

4 BIOCHEMISTRY FOR STUDENTS

log 10 [CH+] = – pH = – 4.50 = 5.50 [CH+] = Antilog 5.50 [CH+] = Antilog 0.5 × antilog 5.00 = 3.16 × 10–5 M. Buffers Buffers are the solutions, which resist changes in pH, when small amount of acid or alkali is added to them. The best buffer is the one which gives the smallest change in pH. Buffers act like shock absorber against the sudden changes of pH. Acetic acid: sodium acetate (CH3COOH; CH3COONa) and carbonic acid: sodium carbonate (H2CO3; NaHCO3) are examples of buffer systems. Physiologic buffers include bicarbonate, orthophosphate and proteins. A buffer is a pair of weak acid and its salt with a strong base or a pair of weak base and its salt with a strong acid. If either free H+ or free OH– are added to a solution of such a pair they will be partially converted to the unionized form. Thus or

B– + H+ HB + OH–

BH H2O + B–

Where HB denotes a weak acid and B– its conjugate base. The combination of a weak acid and the base that is formed on dissociation is referred to as a conjugate pair. Ammonium ion NH+4 is an acid because it dissociates to yield a H+ and NH3 which is conjugate base. Phosphoric acid (H3PO4) is an acid and PO4–3 is a base. NH4+ (acid) H3PO4

H+ + NH3 (conjugate base) 3H+ + PO43¯

The ability to buffer hydrogen ions is more important to the body than the buffering of hydroxyl ions. The most commonly used buffers in the laboratory are: Acetate buffer (Sodium acetate/acetic acid). Phosphate buffer (Na2HPO4/KH2PO4). Citrate buffer (Sodium citrate/citric acid).

BIOPHYSICS

5

Barbitone buffer (Sodium diethyl barbiturate/diethyl barbituric acid). The pH of a buffer solution is calculated by the HendersonHasselbalch equation. Suppose the solution is composed of a weak acid [HA] and its salt with a strong base [BA]. The dissociation of weak acid [HA] and salt [BA] can be represented as follows: HA H+ + A– Weak acid Proton + Conjugate base Conjugate base (A–) is the ionized form of a weak acid [H+] + [A+]

[HA]

+

[BA]

–

[B ] + [A ]

...(1) ...(2)

[HA] dissociates less because it is a weak acid, whereas [BA] dissociates completely because it is a salt of a strong base. Larger the ka, the stronger the acid, because most of the HA will be converted into H+ and A–. Conservely, smaller the ka, less acid will be dissociated and hence weaker the acid. The dissociation constant of equation (1) is represented as: Ka =

[H+ ] [A - ] [HA]

By rearrangement [H+] [A–] = Ka [HA] [H+] =

K a [HA] [A + ]

As the acid [HA] is weak acid, it will be very slightly ionized, and most of it will be present as [HA], whereas the

6 BIOCHEMISTRY FOR STUDENTS

salt [BA] will be highly ionized, the concentration of [A–] can be taken as the total concentration of [BA]. [H+] =

K a [HA] [BA]

Taking logarithm of both sides [HA] [BA]

log [H+]

= log Ka + log

–log [H+]

= –log Ka + log

[BA] [HA]

pH

= pK + log

[BA] [HA]

pH

= pK + log

[Salt] [Acid]

This equation is called Henderson-Hasselbalch equation. If the value of K (the dissociation constant) is known, the pH of a buffer solution of a given composition can be readily calculated. The above equation indicates that the pH of the buffer solution depends on the ratio of the concentrations of the salt and the acid. The buffering power of a mixture of a weak acid and its salt is greatest when the two substances are present in equivalent proportions. Then the buffer has its maximum capacity to absorb either H+ or OH– ions. So that pH is approximately equal to the pK of the acid, i.e. when the acid is half neutralized. [salt] For example [salt] acid log

Therefore

= [acid] = 1

[salt] = log 1 = 0 acid

pH = pK

BIOPHYSICS

7

The effective range of a buffer is 1 pH unit higher or lower than the pKa. The pKa value of most of the acids produced in the body is well below the physiological pH, hence, they ionizes, immediately and add H+ to the medium. The effect of dilution on the pH of a buffer mixture and on the apparent pK of the acid is slight. The pH depends upon the ratio of salt: acid and this ratio is not much affected by dilution. The pH of the buffer solution is determined by the pK and the ratio of salt to acid concentration. Lower the pK value, lower is the pH of the solution; whereas the ratio of salt to acid concentration may vary with no change in pH as long as the ratio remains the same. When the ratio between the salt and the acid is 10:1, the pH will one unit higher than the pKa whereas when the ratio between salt and acid is 1:10 the pH will be one unit lower than the pKa. Maximum buffering capacity occurs ± 1 pH unit on either side of pKa. Buffers are of main importance in regulating the pH of the body fluids and tissues within limits consistent with life and normal function. Many biochemical reactions, including those catalysed by enzymes, require pH control which is provided by buffers. Dissociation constant and pK of acids of importance in biochemistry. Compound

Dissociation constant

Acetic acid Citric acid Lactic acid Pyruvic acid Water Succinic acid

1.74 8.12 1.38 3.16 1 6.46

× × × × × ×

10–5 10–4 10–4 10–3 10–14 10–5

pK 4.76 3.09 3.86 2.50 14 4.19

Q. A mixture of equal volumes of 0.1 M NaHCO3 and 0.1 M H2CO3 shows a pH of 6.1. Calculate the pKa of H2CO3. Ans. Concentration of H2CO3, i.e. acid = 0.1 M. Concentration of NaHCO3, i.e. salt = 0.1 M. Applying Henderson-Hasselbalch equation pH = pK acid + log

[NaHCO 3 ] [H 2 CO 3 ]

8 BIOCHEMISTRY FOR STUDENTS

0.1 0.1 6.1 = pK acid + log 1

6.1 = pK acid + log

pK acid = 6.1

[log 1 = 0]

Q. Phosphate buffers are prepared by mixing together 0.1 M Na2HPO4 and 0.1 M KH2PO4 in different ratios. Calculate the expected pH of the buffer solution prepared by mixing the salt and the acid in the above system in the ratio 2:1 (Given log 2 = 0.30 and pK2 of phosphoric acid 6.7) Ans. Concentration of Na2HPO4 (i.e. salt) = 2 × 0.1 M. Concentration of KH2PO4 (i.e. acid) = 1 × 0.1 M. Applying Henderson-Hasselbalch equation pH = pK phosphoric acid + log = 6.7 + log

[Na 2 HPO 4 ] [KH 2 PO 4 ]

2 × 0.1 1 × 0.1

= 6.7 + log 2 = 6.7 + 0.3 = 7 So the e×pected pH of the buffer solution is 7. Q. You are provided with ample supply of carbonic acid and sodium bicarbonate. How would you prepare a buffer solution of pH 6.1. Give the theoretical basis of the procedure to be followed (pKa of carbonic acid = 6.1). Ans. Applying Henderson-Hasselbalch equation: pH = pK + log

[Salt] [Acid]

pKa of carbonic acid = 6.1 The buffer solution to be prepared should have a pH of 6.1. This can be achieved if the concentration of sodium carbonate and carbonic acid is the same.

BIOPHYSICS

9

So buffer solution of pH 6.1 can be made by mixing equal volume of sodium carbonate and carbonic acid of same concentration. Q. What would be the pH of 100 cm3 of a 0.2 M acetic acid solution to which has been added 10 cm3 of 1.5 M sodium hydroxide. (Given the pK for acetic acid 4.74.). Ans. Before the addition of NaOH, The number of moles of acetic acid present is: 100 = 0.02 M 1000 Also the number of moles of sodium hydroxide present in 10 cm3 of 1.5 M NaOH solution are: 0.2 ×

100 = 0.015 M 1000 Before the start of reaction, the concentration of acetic acid is 0.02 M and that of sodium hydroxide is 0.015 M. When the reaction takes place, i.e. 0.015 M NaOH neutralizes 0.015 M of CH3COOH to form 0.015 M of sodium acetate. After the reaction is over, the concentration of CH3COOH left behind 0.02 M – 0.015 M = 0.005 M. Reaction CH3COOH + NaOH ↔ CH3COONa + H2O Now, applying Henderson-Hasselbalch equation 1.5 ×

pH = pK + log 10

[Acetate] [Acetic acid]

= 4.74 + log 10

0.015 0.005

= 4.74 + log103 = 4.74 + 0.48 = 5.22 Blood Buffers The buffer systems of blood are: 1. Bicarbonate-carbonic acid (BHCO3 : H2CO3)

10 BIOCHEMISTRY FOR STUDENTS

2. 3. 4. 5.

Hemoglobinate-hemoglobin (BHb : HHb) Oxyhemoglobinate-oxyhemoglobin (BHbO2 : HHbO2) Phosphate buffer (B2HPO4 : BH2PO4) Protein buffer (B Protein : H Protein).

The most important buffer of plasma is bicarbonate-carbonic acid system. It is present in high concentration. It is of great importance in the acid-base balance of the extracellular fluid and in the maintenance of the blood pH within normal limits. The bicarbonate system is of prime physiological importance, and acts cooperatively with other buffers. The hemoglobinate-hemoglobin and oxyhemoglobinateoxyhemoglobin buffer, i.e. hemoglobin buffers are of prime importance in the erythrocytes. Hemoglobin actually absorbs 60 percent of the hydrogen ions produced by H2CO3. Hemoglobin is a better buffer than most proteins at pH 7.4 because of relatively high concentration of imidazole group (pKa approximately 7) of the constituent histidine molecules. Deoxyhemoglobin is a better buffer than oxyhemoglobin. The converse is also true, i.e. the hydrogen ions decrease the affinity of hemoglobin for oxygen. Protein and phosphate buffers are of little importance in the blood, i.e. they are the minor buffering systems in the blood. Proteins are present in much higher concentrations in cells than in plasma. They are probably important in buffering H+ ions before their release from cells. But phosphate buffer is of importance in raising the plasma pH through excretion of H2PO¯4 by kidney. It is an important urinary buffer and works cooperatively with the bicarbonate system. Approximate contribution of individual buffers to total buffering in whole blood is given below. Individual buffers Hemoglobin and oxyhemoglobin Organic phosphates Inorganic phosphates Plasma proteins Plasma bicarbonate Erythrocyte bicarbonate

Percent buffering in whole blood 35 3 2 7 35 18

BIOPHYSICS

11

Indicators Indicators are substances which change in color with change in the pH of the solution in which they are present. Indicators are dyes which are weak acids or weak bases and have the property of dissociating in solution. Their ionized form have one color and their unionized form have another color. The color of an indicator solution depends on the relative amounts of its acid and base form present in the solution. An indicator which is a weak acid, is undissociated in acid solution and gives the acid color. In the presence of alkali, it forms a salt which dissociates and displays alkali color. Indicators are used in: 1. Determining the end point in acid-base titrations. 2. Determining pH of solutions. Universal Indicator It is a mixture of a number of indicators which gives a variety of color changes over a wide-range of pH. Some common indicators useful for biological pH range are: Indicators 1. Thymol blue (acid range)

pK pH range solution

Color In acid In alkaline solution

1.65

1.2–2.8

Red

Yellow

–

2.9–4.0

Red

Yellow

3. Methyl orange

3.46

3.1–4.4

Red

Orange Yellow

4. Methyl red

5.00

4.3–6.1

Red

Yellow

5. Phenol red

7.81

6.7–8.3

Yellow

Red

6. Thymol blue (alkaline range)

8.90

8.0–9.6

Yellow

Blue

7. Phenolphthalein

9.70

8.2–10

Colorless Pink

2. Methyl yellow (Topfer’s reagent)

12 BIOCHEMISTRY FOR STUDENTS

OSMOSIS AND OSMOTIC PRESSURE Osmotic flow occurs whenever a semipermeable membrane separates a solution and its pure solvent or between two solutions differing in concentrations. Water molecules pass through the membrane until the concentration on both sides becomes same. Such a movement of solvent molecules from a pure solvent or dilute solution through a semipermeable membrane is called osmosis. Osmotic Pressure Osmotic pressure is the pressure that must be applied on a solution to keep it in equilibrium with the pure solvent when the two are separated by semipermeable membrane or osmotic pressure is the force required to oppose the osmotic flow.

Hypertonic solutions: If the osmotic pressure of the surrounding solution is high, water passes from the cell to the stronger solution outside, this causes the cell to shrink away. Isotonic solutions: If external solution has the same osmotic pressure, no flow of water takes place and hence no effect upon the cell protoplasm is observed. Hypotonic solutions: If the osmotic pressure of the surrounding solution is low, water passes into the cell from the surrounding, the cells become turgid and rupture. Van’t Hoff’s law of osmotic pressure: 1. The osmotic pressure of a solution is directly proportional to the concentration of the solute in the solution. 2. The osmotic pressure of a solution is directly proportional to the absolute temperature. Thus indirectly they follow Boyle’s and Charle’s Law. Osmotic pressure is given by the formula. π = CRT where π = Osmotic pressure C = Concentration in moles per liter R = Gas constant T = Absolute temperature

BIOPHYSICS

13

Osmotic pressure is dependent upon the number of dissolved particles (i.e. on concentration) and is independent of the size or weight of the particle. According to the law of osmotic pressure, 1 molar solution exerts an osmotic pressure of 22.4 liters at 0ºC. The osmotic pressure of substances which ionizes is given by the formula. π = i CRT where i the isotonic coefficient is given by: i = 1 + α (n–1) α = degree of ionization n = number of ions obtained on ionization The value of i, depends upon the degree of dissociation of the electrolyte, which varies from one electrolyte to another. It increases as the dilution increases and depends upon the number of ions formed. Since osmotic pressure is proportional to the total number of solute particles in solution, the substances which ionize, will have the higher osmotic pressure as compared to those substances which do not ionize. The osmotic pressure exerted by colloidal solutions is always less as compared to that of crystalloids of similar concentrations in gram per liter because the magnitude of osmotic pressure depends upon number of particles present in unit volume of the solution. Solutions that exert the same osmotic pressure are called isomotic. The osmotic pressure of 1 M NaCl will be double, as compared to the osmotic pressure of 1 M sucrose or glucose solution because each molecule of NaCl on ionization gives two ions, i.e. Na+ and Cl– ions and each ion will exert the respective osmotic pressure. The unit of osmotic pressure is osmol or milliosmol. An osmolar solution is defined as one exerting the osmotic pressure of a molar solution of a nondissociated solute in one liter of solution. Thus the number of osmoles of a undissociated substance in a liter of solution would be the weight in grams divided by its molecular weight. The milliosmolar concenwhere

14 BIOCHEMISTRY FOR STUDENTS

tration of glucose in a sample of plasma containing 90 mg per 100 ml therefore would be: 90 mg per 100 ml × 10 = 5 milliosmol per liter 180 (Mol. wt of glucos e)

For nonelectrolytes such as glucose or sucrose, 1 millimol is equal to 1 milliosmol. For electrolytes such as NaCl, one millimol of NaCl is equivalent to 2 milliosmol (Na+ and Cl¯). Q. 1 Molar solution of glucose has an osmotic pressure of 22.4 atmosphere at 0ºC. Calculate the osmotic pressure of 0.1 M sucrose and 0.1 M NaCl at the same temperature. Assume 100% dissociation of NaCl. Ans. 1 molar solution exerts an osmotic pressure of 22.4 atmosphere. So 0.1 Molar solution will exert an osmotic pressure of 2.24 atmosphere. So 0.1 M sucrose will have an osmotic pressure of 2.24 atmosphere. In case of sodium chloride, each molecule of NaCl on dissociation gives Na+ ions and Cl– ions. Each ion, i.e. Na+ and Cl– will exert an independent osmotic pressure. Also the dissociation of sodium chloride is 100%. So 0.1 M solution of NaCl will exert an osmotic pressure of 2 × 2.24, i.e. 4.48 atmospheres. Q. Calculate the osmolarities of: i. 0.1 M NaCl solution ii. 0.1 M sucrose solution Ans. The term milliosmol is used in connection with osmotic pressure. 0.1 M solution of NaCl will have an osmotic pressure of 0.1 × 2 = 0.2 milliosmol (because each molecule of sodium chloride on ionization gives two ions). Whereas 0.1 M sucrose will have an osmotic pressure of 0.1 milliosmol. Milliequivalent One milliequivalent is one thousandth of an equivalent and is the same as millimol as long as the valency is one.

BIOPHYSICS

15

For valence 1; 1 millimol = 1 milliequivalent For valence 2; 1 millimol = 2 milliequivalent For valence 3; 1 millimol = 3 milliequivalent How to calculate millimols? millimol =

milligrams per liter Formula weight

Example: 78 mg of K+ ions per liter = 78/39, i.e. 2 millimols = 2 milliequivalent = 2 milliosmols Whereas 100 mg of Ca++ per liter

= 100/40, i.e. 2.5 millimols = 2.5 milliosmols = 5 milliequivalent

222 mg of CaCl2 per liter

Ca = 40,

2Cl

–

= 222/111, i.e. 2 millimols of CaCl2 = 6 milliosmols. = 2 × 35.5 = 71

Gibbs Donnan Equilibrium Gibbs Donnan equilibrium is concerned with the distribution of electrolytes in systems separated by membranes which are impermeable to certain components. This resultant unequal distribution of diffusible ions due to the presence of nondiffusible ions on one side of the membrane is called Gibbs Donnan Effect. Example: Consider a semipermeable membrane separating a solution of NaCl and Protein (NaR). The membrane is permeable to Na+ and Cl– but not to R–. Na+ R–

Na+ Cl–

In the beginning (A)

Na+ Na+ – R Cl– Cl – At equilibrium (B)

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When the equilibrium is attained, the product of concentrations of diffusible ions (Na+ and Cl–) on one side of membrane is equal to the product of concentrations of same ions on the other side, i.e. (Na+)(Cl–) > (Na+)(Cl–) The concentration of diffusible positive ion is greater on the side of membrane containing nondiffusible ion, i.e. [Na+]1 > [Na+]2 Donnan effect is of physiological significance in biological systems involving ion exchanges across permeable membranes when the fluid on one side of the membrane contains a nondiffusible component. This results in difference of concentration of diffusible ions which leads to junction potential across the membrane, which is a driving force for most of the body reaction. Donnan effect is also involved in absorption, secretion and maintenance of different electrolyte concentrations between various compartments of the body. COLLOIDS Graham classified substances into: 1. Crystalloids: Substances which pass through parchment or animal membrane. 2. Colloids: Substances which do not pass through parchment or animal membrane. But nowadays, the size of the molecule or particle determines whether they will form crystalloidal or colloidal solutions. According to modern concept. True solution

Colloidal solution

Suspension solution

where the size (diameter) of the particle is less than 1 mμ

where the size is between 1-20 mμ

where the size is more than 200 mμ

Properties of Colloidal Solutions 1. Dialysis: The process of separation of crystalloids from colloids by diffusion through a membrane by osmotic force

BIOPHYSICS

2.

3.

4. 5.

17

is called dialysis. Dialysis has an important application in medicine in the artificial kidney. This device is inserted into the patient’s circulation and diffusible material particularly urea passes out from the blood substituting for the action of the faulty kidneys. As the size of the colloidal particle is large, few particles are present in small concentration, the osmotic pressure of the colloidal solution will be very small. This is of prime importance in driving the passage of water and other substances through cell membranes. Precipitation: Colloids possess net charge at the surface which arises from ionisable groups on the particle surface and also from absorption of ions and can be precipitated by neutralizing the charge. Brownian motion. Tyndall effect. SURFACE TENSION

The force with which the surface molecules are held in a solution is called surface tension. Some substances such as bile salts have the property of lowering the surface tension of the medium in which they are present. This effect is used in the absorption of fats from the intestine. Other properties of surface tension are formation of drops of liquids falling through air; rise of liquid in a capillary tube and formation of meniscus at the surface of liquids. Surface tension decreases with increase in temperature. Role of Surface Tension Substance which lower the surface tension becomes concentrated in the surface layer whereas substances which increase surface tension are distributed in the interior of the liquid. Soaps, oils, proteins and bile acids reduce the surface tension of water, while sodium chloride and inorganic salts increase the surface tension. Surface tension leads to better adsorption.

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ABSORPTION Certain substances have the power of making water insoluble substances soluble in water without any apparent chemical alteration of the dissolved substance. The substances having such quality are called hydrotropic substances. Among the insoluble substances which are brought into the solution are fats, phospholipids, sterols, calcium carbonate, magnesium phosphate, etc. Substance which bring about the solubility are cholic acids, benzoic acid, hippuric acid, soaps of higher fatty acids, etc. The biological importance of the solution of an insoluble substance in hydrotropic substances lie in the fact that the substances so dissolved are diffusible through membranes. VISCOSITY Viscosity of a liquid is the resistance to flow. Viscosity of blood is 4.5 times more than water. Viscosity of blood is lowered in anemia, nephritis, leukemia, malaria, diabetes mellitus, jaundice, whereas excessive sweating and shock leads to increase of blood viscocity.

CHAPTER

2

Chemistry of Carbohydrates CARBOHYDRATES

Carbohydrates are defined as the aldehydic or ketonic derivatives of polyhydroxy alcohols and their polymers having hemiacetal glycosidic linkages. The general formula for carbohydrates is Cn(H2O)n. Carbohydrates are the main source of energy in the body. Brain cells and RBCs are exclusively depend on carbohydrates (glucose) as the energy source. The sugar is a carbohydrate and is sweet to taste, soluble in water and chars on heating. Glucose (Grape sugar), fructose (fruit sugar), sucrose (cane sugar), lactose (milk sugar), and maltose (malt sugar) are few examples of sugar. All sugars are carbohydrates but all carbohydrates are not sugars. Glycogen and inulin are carbohydrates but not sugars. FUNCTIONS OF CARBOHYDRATES 1. Provides energy, i.e. as major source of energy to the body. Their calorific value is 4 kcal per gm. 2. As structural components of membranes. 3. As structural basis for DNA and RNA (Ribose/Deoxyribose). 4. As structural basis for nucleosides and nucleotides. 5. As source of carbon skeltons for some amino acids. 6. As basis of some intracellular messenger systems. CLASSIFICATION OF CARBOHYDRATES Monosaccharides Monosaccharides consists of single polyhydroxy aldehyde or ketone unit which cannot be broken down to simpler substances on acid hydrolysis. They are also called simple sugars. Monosaccharides are further divided into: i. Aldoses, i.e. Aldo sugars ii. Ketoses, i.e. Keto sugars.

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Aldoses Monosaccharides containing aldehydic group as the functional group are called aldoses. They are classified according to the number of carbon atoms present. Monosaccharides containing three to seven carbon atoms are called trioses, tetroses, pentoses, hexoses and heptoses respectively. Trioses : D-glyceraldehyde (aldotriose) Dihydroxy acetone (ketotriose) Tetroses : D-Erythrose (aldotetrose) Pentoses : D-Xylulose (ketopentose) : D-Ribose (aldopentose) : D-Deoxyribose (aldopentose) : D-Xylose (aldopentose) : D-xylulose (aldopentose) Hexoses : D-Glucose, D-Galactose, D-Mannose (aldohexose) : D-Fructose (ketohexose) Structures of Erythrose, Ribose, Glucose, Galactose, Mannose are:

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21

Ketoses Monosaccharides containing ketonic group as the functional group are called ketoses. Examples: Xylulose, Ribulose, Fructose, etc.

Stereochemistry The presence of asymmetric carbon atoms (an asymmetric carbon atom is one to which four different atoms or groups are attached) in the compound results in the formation of isomers of that compound. The number of isomers of a compound depends on the number of asymmetric carbon atoms and is given by 2n, where n indicates the number of asymmetric carbon atoms in that compound. If the hydroxyl group on the highest asymmetric carbon atom or on the penultimate carbon atom is on the right hand side, than the compound will belong to D-Series. If the hydroxyl group is on the left side, than the compound will belong to LSeries.

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The D-and L-forms of glucose are given below:

Two compounds that resemble each other but are different because their carbons are asymmetric. The relationship exhibited by each compound is called stereoisomerism and the two compounds are called stereoisomers or enantiomorphs. Stereoisomers are those compounds which have the same composition but differ in spatial arrangements. Carbohydrates exhibit the property of optical activity and exist as optical isomers. Glucose with four asymmetric carbon atom will have 24, i.e., 16 isomers. 8 of these isomers will belong to D-series and other 8 to L-series.

(Where X denotes that particular carbon atom is asymmetric).

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In the open chain structure of D-glucose, C2, C3, C4, and C5 are the asymmetric carbon atoms. But in nature, D-glucose exists in 32 stereoisomers, i.e. 32 isomers of D-glucose has been isolated. The 32 isomers can be best explained if there is one more asymmetric center in the D-glucose. This is possible if glucose exists in ring or cyclic structure. The cyclic structure involves the formation of hemiacetal linkage between aldehyde group (i.e. C1) and hydroxyl group at C4. In the process, a new asymmetric centre C1 is created at glucose. In the ring form of D-glucose, C1, C2, C3, C4, and C5 are asymmetric and will have 25, i.e., 32 stereoisomers. During the process of cyclization a six membered ring consisting of five carbon atoms and an oxygen atom is formed in case of glucose. This ring structure is also called pyranose structure.

Similarly a five membered ring consisting of four carbon atoms and an oxygen atom is formed in case of fructose. This ring structure is also called furanose structure.

24 BIOCHEMISTRY FOR STUDENTS

The planar formula of sugars is also called Fischer formula and the ring formula is called Haworth formula.

Epimers: Carbohydrates that differ in their configuration about a specific carbon atom other than the carbonyl carbon atom are called epimers.

Glucose and galactose are epimers as they differ in their configuration at C-4 carbon atom. Similarly, glucose and mannose are epimers as they differ at C-2 carbon atom. The process of interconversion of glucose and galactose is known as epimerization. In glucose, the hydroxyl group at C-4 is on the right hand side whereas in galactose, the hydroxyl group at C-4 is on the left hand side.

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25

Anomers: Carbohydrates that differ only in their configuration around the carbonyl carbon atom are called anomers. The carbonyl carbon atom is called the anomeric carbon atom. α-D-glucose and β-D-glucose are the anomeric forms of D-glucose. In α-D-glucose, the hydroxyl group at C-1 (i.e. carbonyl carbon atom) is on the right hand side whereas in β-D-glucose, the hydroxyl group at C-1 is on the left hand side.

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Anomeric form arises as a result of cyclization or ring formation. During the process of cyclization, the C-1 carbon atom which is symmetrical in the open chain formula of glucose is converted into asymmetric carbon atom. The presence of asymmetrical carbon atom give rise to optical activity. When a beam of plane polarized light is passed through a solution of carbohydrates, it will rotate the light either to left or to the right. Depending upon rotation, molecules are called dextrorotatory (+) or (d), levorotatory (–) or (l). A compound that rotates the plane of polarized light in a clockwise direction is said to be dextrorotatory (+), whereas that which rotates the plane of light in a anticlockwise direction is said to be levorotatory (–). Amino Sugars The amino sugars occurring most frequently are glucosamine and galactosamine. They occur as N-acetyl compounds.

Glucosamine is present in chitin, shells of insects and mammalian polysaccharides whereas galactosamine is present in polysaccharides of cartilage and chondroitin. Reactions of Monosaccharides 1. 2. 3. 4.

Action of acids Mutarotation Reducing property Osazone formation

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5. 6. 7. 8.

27

Action of dilute alkali Oxidation Reduction Glycoside formation.

Action of Acids This is a general test for carbohydrates. Monosaccharides on treatment with concentrated sulphuric acid undergoes dehydration to give furfural or furfural derivatives which on condensation with α-naphthol yield a violet or purple colored complex. Pentoses yield furfural whereas hexoses yield 5-hydroxy furfural.

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Mutarotation Mutarotation is defined as the change in specific rotation of optically active solution without any change in other properties. When glucose is dissolved in water, the optical rotation of the solution gradually changes and attains an equilibrium value. This change in optical rotation is called mutarotation. Mutarotation occurs due to the cyclization of open chain form of glucose into α or β form with equal probability. This α and β cyclic form of glucose have different optical rotations. This is because, the α and β form are not mirror images of each other. They differ in configuration about the anomeric carbon (C1) but have the same configuration at C2, C3, C4, and C5 asymmetric carbons. These cyclic forms are in equilibrium with open chain structure in aqueous solution. Such a change from a single form to an equilibrium mixture that includes its other form is called mutarotation. +112o α-D-glucose

+52-5o Equilibrium mixture contains α, β and open chain forms

+19o β-D-glucose

α-form 36%, β-form 63% and open chain form 1%. The predominance of the β-form in aqueous solution is due to its more stable conformation relative to the α-form. Biologically this change is catalyzed by the enzyme, mutarotase.

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29

In aqueous solution, many monosaccharides behave as if they have one more asymmetric center than is given by open chain structure. Ring formation involves the formation of internal hemiacetal linkage between the aldehyde group, i.e. C-1 and the hydroxyl group at C-5 and a new asymmetric carbon at C1 is created in glucose. In this cyclic form, there are now five asymmetric carbon atoms (i.e. C-1, C-2, C-3, C-4, C-5) which best explains about the existence of 25, i.e. 32 isomers of glucose.

Reducing Property Monosaccharides by virtue of free aldehydic or ketonic group in their structure, i.e. presence of free anomeric carbon atom, reduces certain heavy metallic cation, e.g. Cu++ ions in alkaline solution at high temperature. So all the reducing sugars will give Benedict’s qualitative test and Fehling test positive. The reaction is as follows: The color of the solution or precipitate gives an approximate

(rough) amount of reducing sugars present in the solution. Green color......up to 0.5% (+) Green precipitate.....0.5-1% (++) Green to yellow precipitate.....1.0-1.5% (+++) Yellow to orange precipitate.....1.5-2.0% (++++) Brick red precipitate....more than 2%

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Benedict’s qualitative reagent contains cupric sulfate, sodium carbonate and sodium citrate whereas Fehling solution contains cupric sulfate, sodium hydroxide and sodium potassium tartrate (Rochelle salt). Sodium citrate in Benedict’s reagent and sodium potassium tartrate (Rochelle’s Salt) in Fehling solution prevent the precipitation of cupric hydroxide or cupric carbonate, by forming a deep blue soluble slightly dissociated complexes with the cupric ions. These complexes dissociate sufficiently to provide a continuous supply of readily available cupric ions available for oxidation. Benedict’s qualitative reagent is preferred above Fehling solution because it is more stable. Also traces of sugar which is destroyed by the strong alkali of Fehling solution is not destroyed by Benedict’s reagent.

Osazone Formation Reducing sugars can be distinguished from one another by phenylhydrazine test when characteristic osazones are formed. These osazones have characteristic crystal structures, melting point, precipitation time and show different crystalline forms under a microscope and hence, are valuable in the identification of reducing sugar. Glucose, fructose and mannose give the same osazones and hence, they cannot be differentiated from each other by this test. In the osazone formation only first two carbon atoms, i.e. C-1 and C-2, take part in the reaction. So reducing sugars which differ in their configuration at C-1 and C-2 and have rest of the structure same, i.e. C-3, C-4, C-5 and C-6 have the same configuration, give the same osazones because during osazone formation, the structural dissimilarity at C-1 and C-2 disappears and the rest of the molecule structure is the same. Three molecules of phenylhydrazine are required to produce one molecule of osazone.

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31

The formation of osazones of glucose is explained below.

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Fructose reacts with phenylhydrazine in a similar manner.

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33

Glucose osazone, fructose osazone and mannose osazone are identical with respect to its crystal structure and chemical structure. Glucose, fructose and mannose give the needle shape osazones whereas maltose gives sunflower and lactose gives cotton ball shape osazones.

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Appearance of yellow crystals takes place. Observe the shape of crystals under microscope.

Lactose (Cotton Ball) Maltose (Sunflower) Osazone of maltose and lactose

Action of Dilute Alkali Monosaccharides on treatment with dilute alkali undergo a variety of molecular transformation through enediol formation. The enediols of sugars are good reducing agents and form the basis of reducing action of sugars in alkaline medium. When glucose is treated with dilute alkali for several hours, the resulting mixture obtained contains both fructose and mannose in addition to glucose. A similar mixture of same sugars is obtained with any of the other two sugars. This interconversion of related sugars by the action of dilute alkali is termed as Lobry de Bruyn-van Ekenstein rearrangement (see page 36 for reaction). Whereas sugars on boiling with strong alkalis are caramelized to give yellow to brown resinous product. That is the reason why Benedict’s reagent containing sodium carbonate is preferred to Fehling solution containing sodium hydroxide.

Oxidation Aldoses are oxidized under variety of conditions to the following: i. Aldonic acid: Whereby the first carbon atom (C-1) is oxidized to carboxyl group only. The rest of the molecule structure remains unaffected. ii. Uronic acid: Whereby the terminal carbon atom is oxidized to carboxyl group only. The first carbon atom, i.e. aldehydic group and the rest of the molecular structure

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35

remains unaffected. Uronic acid derivatives are particularly important in detoxification process, i.e., bilirubin is excreted as bilirubin diglucuronide. Besides this, D-glucuronic acid, D-galactouronic acid, D-mannouronic acid, L-induronic acid are important components of polysaccharides. iii. Aldaric or saccharic acid: Whereby both the first carbon atom, i.e. aldehydic group and the terminal carbon atom, i.e. primary alcoholic group are oxidized to carboxyl group. Galactose undergoes oxidation to form a dicarboxylic acid, mucic acid. This reaction is often important in the identification of galactose.

Example: The oxidation products of glucose under different conditions are given on Page 37. Glucose Oxidase: The substrate for glucose oxidase is βD-glucopyranose. Blood glucose which is an equilibrium mixture of α- and β-anomers of D-glucose is qualitatively determined by the formation of hydrogen peroxide by the reaction (P-39).

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37

Two very important uronic acids occuring in carbohydrates are D-glucuronates and L-iduronate (from hexose idose). The only difference between these two molecule is that the carboxyl group is above the ring for D-glucuronate and below the ring for L-iduronate.

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This requires that the α-D-glucopyranose be rapidly isomerized by mutarotation into the β-D-isomer. This reaction is fast without catalyst. Q. A reducing carbohydrate gives a positive reaction with Barford’s test and mucic acid crystals on oxidation. Give the structure of that carbohydrate. Would it exhibit property of mutarotation. If so, what products are formed at equilibrium. Ans. Since Barford’s test is positive. It indicates that reducing carbohydrate is monosaccharide. Also mucic acid crystals are obtained on oxidation suggesting that the given reducing carbohydrate is galactose as it is the galactose which on oxidation gives mucic acid crystals.

D-galactose will show mutarotation due to the cyclization of open chain form of D-galactose into α- and β- form with equal probability. The products at the equilibrium are:

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39

Reduction Glucose on reduction gives sorbitol. Whereas fructose on reduction gives a mixture of sorbitol and mannitol. Mannose gives mannitol, galactose is reduced to dulcitol and ribose to ribotol.

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Fermentation: Fermentation is the process of breakdown of complex organic substances into smaller substances with the help of enzymes. Glucose is fermented to ethyl alcohol and carbon dioxide by yeast. Hence this process is called alcoholic fermentation as alcohol is produced. Glycosides Formation Glycosides are sugar derivatives in which hydrogen of the hydroxyl group of hemiacetal or hemiketal form of the sugar is replaced by an organic moiety. A molecule of water is eliminated when this reaction takes place. Glycosides are not reducing sugars and do not show mutarotation. If the organic moiety is derived from another monosaccharide, the product formed is disaccharide. If the organic moiety is a noncarbohydrate, then it is called aglycone. Aglycone: The noncarbohydrate portion of the glycoside is called the aglycone or aglucone. Glycosides do not reduce alkaline copper sulphate because sugar group is combined, i.e. aldehyde group is converted to an acetal group. Glycosides = Carbohydrate + Carbohydrate part or noncarbohydrate part (aglycone) Examples Cardiac glycosides = Carbohydrate + Digoxin or digitoxin (aglycone) Indican = Carbohydrate + Indoxyl (aglycone) Amygdalin = Carbohydrate + Benzaldehyde (aglycone) OLIGOSACCHARIDES Oligosaccharides are arbitrarily defined as carbohydrates that contains two to ten monosaccharide units per molecule joined by glycosidic linkages. On hydrolysis they yield monosaccharides. Depending upon the number of constituent monosaccharide units, the oligosaccharides are called disaccharides, trisaccharides, etc.

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Oligosaccharides are reducing sugars if one of the carbonyl group is free (not involved in glycosidic linkage). The reducing power of carbohydrate decreases as the number of their sugar components increases. Disaccharides Disaccharides consist of two monosaccharides joined by a glycosidic linkage. The most common and important disaccharides are maltose, Lactose and Sucrose. Maltose and lactose are reducing disaccharides whereas sucrose is nonreducing disaccharide. In general, the properties of disaccharides are similar to those of monosaccharides. Reducing disaccharide sugars are not as reducing agents as monosaccharide because of the lower ratio of reducing groups to carbon atoms. Maltose Maltose consists of two molecules of D-glucose joined by α (1,4)-glycosidic linkage. The anomeric carbon of one glucose molecule is joined to the C-4 carbon of the second glucose molecule. The anomeric carbon of the second glucose molecule is free. So maltose is a reducing disaccharide.

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Maltose or malt sugar does not occur in free state but is formed as an important transitory intermediate product of the digestion of starch and glycogen. Maltose reduces heavy metallic ions in alkaline solution (e.g. Benedict’s reagent), undergoes mutarotation and forms sunflower crystals of maltosazone with phenylhydrazine. Lactose Lactose consists of galactose and glucose joined by β (1,4)glycosidic linkage. The anomeric carbon of D-galactose is joined to 4-carbon of D-glucose. The anomeric carbon of Dglucose is free, so lactose is a reducing disaccharide. Lactose is glucose galactoside. Lactose or milk sugar is an animal disaccharide and is present to the extent of 5% in milk only. It is synthesized in mammary gland and during lactation may appear in the urine.

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Lactose on treatment with concentrated nitric acid gives mucic acid crystals. Lactose reduces Benedict’s reagent, undergoes mutarotation and forms cotton ball lactosazone crystals with phenylhydrazine. Sucrose Sucrose is a non-reducing disaccharide. Sucrose consists of glucose and fructose joined by α(1) →β(2) glycosidic linkage. The anomeric carbon (C-1) of glucose molecule in α configuration is linked to anomeric carbon (C-2) of fructose in β configuration. So sucrose is a nonreducing disaccharide as both the reducing groups of glucose and fructose are linked together and hence not available for reduction. Sucrose or sugar cane is a plant disaccharide and is present in high concentration in sugar cane and sugar beet. Sucrose is used for sweetening purpose.

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Sucrose does not reduce Benedict’s reagent, does not show mutarotation and does not form osazone with phenylhydrazine. Invert Sugar Sucrose on hydrolysis yields equimolecular amounts of glucose and fructose. Since this mixture is levorotatory whereas the original sucrose is dextrorotatory, the process is known as inversion because of the inversion of the sign of rotation, and the mixture of glucose and fructose obtained is called as invert sugar. H+ Sucrose Glucose + Fructose (+65.5) (+52.7) (–92) Honey contains large amount of invert sugar. Isomaltose Isomaltose, a disaccharide is derived from the branch point of starch. Isomaltose has α (1→ 6)-D-glucosidic linkage to a second D-glucose residue.

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POLYSACCHARIDES Polysaccharides are the polymers of monosaccharide units which are joined in linear or branched chain fashion by glycosidic linkages. Polysaccharides contain a large number of sugar components per free carbonyl group. In a branched polysaccharides, there is only one reducing end and multiple nonreducing ends. Thus these free carbonyl groups are not sufficiently potential to reduce the Benedict’s Reagent, etc. By convention polysaccharides are given names ending in— an attached to the particular monosaccharide that make up the polymer. Thus a name for a polysaccharide in general is glycans from glucose. Examples are mannans, xylans and arabans which are polymers of mannose, galactose, xylose and arabinose. Polysaccharides have two important biological functions. 1. As storage form of fuel (i.e., glycogen of animal origin and starch of plant origin). Glycogen and starch are both storage form of glucose; glycogen is used by animals to store glucose and starch is used by plants. 2. As structural components, e.g. Cellulose. The structural polysaccharides have β-linkage and the storage polysaccharides have an α-linkage. The β-linkage keeps the molecular linear whereas α-linkage tends to fold the molecule, forming a gloublar structure then linear one. Polysaccharides can be divided into two groups: a. Homopolysaccharides b. Heteropolysaccharides. Homopolysaccharides They contain only one type of monosaccharides as the repeating unit and on hydrolysis gives only one type of sugar. Example: Starch, cellulose, glycogen, dextrins, etc. Starch Native starch is a mixture of two polysaccharides. a. Amylose b. Amylopectins.

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Amylose Amylose is a linear unbranched molecule in which D-glucose units are linked by α–(1→4) glycosidic linkages. It is water soluble and gives blue color with iodine.

Amylopectin Amylopectin is a branched chain molecule in which D-glucose units in addition to α-(1,4) linkages are branched by α-(1,6) glycosidic linkages. This branching occurs on an average of 24 to 30 D-glucose units. It is water insoluble and gives violet color with iodine.

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Starch is a nonreducing polysaccharide, tasteless substance and gives blue color with iodine. Starch on hydrolysis with dilute mineral acids, i.e. with hydrochloric acid gives glucose only. Action of amylases on starch: Amylases are hydrolytic enzymes which hydrolyze polymers of glucose containing α-(1 → 4) glycosidic linkages, Amylases are of two types: 1. α-Amylases. 2. β-Amylases. α-amylases are present in saliva and pancreatic juice. They act on starch, hydrolyzing α-(1,4) glycosidic linkages in a random manner to yield glucose, free maltose and smaller units of starch called starch dextrins. These starch dextrins contain the original α-(1,6) glycosidic linkages. α-amylase cannot hydrolyze the α-(1,6) linkages at the branched point of amylopectins. The α-amylases are activated by chloride ions. β-amylases present in barley malt, cleave successive maltose units beginning from nonreducing ends of starch to give maltose. β-amylase yield only maltose with amylose and smaller branched polysaccharides, known as limit dextrins, as well as maltose with amylopectin. β-amylases also cannot hydrolyze α-(1,6) linkages at the branched point of amylopectin. Cellulose Cellulose is a linear polymer of D-glucose units joined together by β–(1,4) glycosidic linkages. On partial hydrolysis, cellulose yields β-1,4 disaccharide cellobiose instead of maltose. Cell-ulose is water insoluble, nonreducing and gives no color with iodine. Unlike starch and glycogen which are readily digested, cellulose cannot be utilized for energy purposes by human beings, because the enzyme which cleavage β-(1,4) linkage is missing in the gastrointenstinal tract and hence, merely provide bulk to the diet. Cellulose is present in plant leaves, stems, and outer coverings of fruits and vegetables. Cellulose

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is a component of fiber (nondigestible carbohydrate) in the diet. Cellulose is present in plant leaves stems and outer coverings of fruits and vegetables. Cellulose aids intestinal mobility and acts as an stool softener and reduces bowel cancer. The nutrition value of cellulose is nil. Celluloses are the most abundant organic compound on earth. Celluloses are the major components of plants comprising 20 to 45% of this cell wall mass.

Glycogen Glycogen is the carbohydrate reserve of the body. Glycogen is also called animal starch, because it serves as nutritional reservoir in animal tissues. Glycogen is a highly branched chain molecule in which glucose unit in addition to linear α-(1,4) linkages are also linked by α-(1,6) at the branched point. This branching repeats after every 8-10 glucose units. Glycogen is water soluble and has no reducing property. It gives red color with iodine. Glycogen is stored in liver and muscle. About three-fourth of all the glycogen in the body is stored in muscle. Difference between starch and glycogen. 1. Starch is of plant origin whereas glycogen is of animal origin. 2. Glycogen is much more branched than the starch. In starch, the branching is after every 24 to 30 glucose units, whereas in glycogen, the branching is after every 8 to 10 glucose units. 3. Starch gives blue color with iodine solution whereas glycogen gives red color. Dextrins They are the partial hydrolytic products of starch by α-amylase, β-amylase and acids. Dextrins formed from amylases have

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unbranched chains while those formed from amylopectins are branched. All dextrins have free sugar group and accordingly reduce alkaline copper sulphate solution. HETEROPOLYSACCHARIDES Heteropolysaccharides are made up of mixed disaccharides repeating units and on hydrolysis gives a mixture of more than one product of monosaccharides and their derivatives of amino sugars and sugar acids. They are the essential components of the tissues where they are present in combination with proteins as mucoproteins. They are also called mucopolysaccharides or glycosamino glycans (CAG). The other suitable name for such heteropolysaccharides is Glycosaminoglycan or CAG. Glycosaminoglycans are unbranched polysaccharides consisting of repeating dissaccharide units comprising a sugar linked to either N-acetylglucosamine or N-acetylgalactosamine. They can be divided into: 1. Neutral mucopolysaccharides 2. Acidic mucopolysaccharides. Acid mucopolysaccharides are present in connective tissues. They contain hexosamine as the repeating disaccharide unit. The repeating structure of each disaccharide contains alternate 1,4 and 1,3 linkages. The most common CAGs are: Hyaluronic Acid Hyaluronic acid is present in the connective tissues, synovial fluid and vitreous fluid in combination with proteins. It is an unbranched polymer. The repeating disaccharide is made up of D-glucuronic acid and N-acetyl D-glucosamine. The monosaccharide subunits are linked by β-(1,4) and β-(1,3) glycosidic linkages. Glc UA-β(1 → 3) – Glu NAc connected by β(1 → 4) linkages.

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On acid hydrolysis it gives an equimolar quantities of glucuronic acid, glucosamine and acetic acid.

Hyaluronates form viscous lubricants of joints and gel like substance inside the eyes-vitreous humor. Heparin Heparin is glucosaminoglycans. Heparin is an acidic mucopolysaccharide in which both the amino and the hydroxyl groups are combined with sulphuric acid, which causes it to be slightly acidic substance. Heparin is present in liver, lungs, thymus, spleen and blood. Heparin is blood anticoagulant. Heparin contains D-glucosamine, D-glucuronic acid or L-iduronic acid as the repeating disaccharide units. The glucosidic linkage is α(1,4) involving the glucuronic acid anomeric carbon hydroxyl with hydroxyl group at C-4 of glucosamine.

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Chondroitin Sulfates They are present in connective tissues and serve as a structural material such as cartilage, tendons and bones. Chondroitin sulfates are sulfated polysaccharides. Chondroitin sulfate is galacto aminoglycans. The acid hydrolysis of chondroitin sulfate yield D-galactose, D-glucuronic acid, acetic acid and sulfuric acid.

Sialic Acids Sialic acids are N-acetyl derivatives of neuraminic acid and are widely distributed in tissues such as mucins are present in blood group substances.

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Neuraminic acid is a condensation product of pyruvic acid and mannosamine. Examples

Repeating units

Hyaluronic acid Chondroitin Chondroitin-4sulfate (Chondroitin sulfate A) Heparin

Glucuronic acid; N-Acetyl glucosamine Glucuronic acid; N-Acetyl galactosamine Glucuronic acid; N-Acetyl galactose-4-sulphate Glucosamine-6-SO4; glucuronic acid-SO4; iduronic acid

Other CAGs 1. Chondroistin sulfate and dermatan sulfate are galactosamine glycam. 2. Heparin sulfate, heparin and keratan sulfate are glucosamine glycam. Mucoproteins and Glycoproteins If the carbohydrate associated with protein is greater than 4%, then the complex protein is called mucoprotein. If the carbohydrate content is less than 4%, then is called glycoprotein. Plasma α1 and α2 globulins are glycoproteins. Blood Group Substances They are water soluble, high molecular weight substances, made up of polysaccharides and proteins. They are present in saliva, gastric mucin, erythrocyte membranes, etc. The immunological specificity resides in oligosaccharide part. The residues present in the oligosaccharides are L-fucose, D-galactose, N-acetyl-D-galactosamine and N-acetyl glucosamine.

CHAPTER

3

Chemistry of Lipids

According to Bloor, lipids are defined as a group of naturally occurring substances consisting of the higher fatty acids, their naturally occurring compounds and substances found naturally in association with them. It includes a wide variety of substances with different structures. They are insoluble in water but are soluble in so-called fat solvents such as ether, acetone, chloroform, benzene, etc. Associated with them are various fat soluble, non-lipid substances which includes carotenoid pigments and certain vitamins, i.e. vitamins A, D, E and K. Lipids are widely distributed throughout both plant and animal kingdom and are essential constituents of cell membrane. Fats are said to be protein sparing because their availability in the diet reduces the need to burn proteins for energy. Lipids have several important biological functions. 1. They serve as the reservoir of energy because of their: a. High energy content. The calorific value is 9 kcal/gm as compared to carbohydrates which have calorific value of 4 kcal/gm. b. Storage in concentrated form in water free state (anhydrous) in the tissues as compared to carbohydrates which are highly hydrated and cannot be stored in such concentrated form. 2. As structural components of cell membranes. 3. As transport forms of various metabolic fuel. 4. As protective coating on the surface of many organs such as kidney, against injury. 5. To facilitate the absorption of the fat soluble vitamins A, D, E and K.

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Dietary fat can be divided into two types: a. Visible fat or fat consumed as such, e.g. butter, oils, ghee. b. Invisible fat or fat present as part of other foods items, e.g. egg, fish, meat, cereal, nuts, etc. Classification and Functions of Lipids Classification 1. Fatty lipids 2. Triglycerides 3. Phospholipids 4. Sphingolipids 5. Ketone bodies

Functions Metabolic fuel, building block for other lipids Fatty acid storage, transport Membrane structure, storage of arachidonic acid Membrane structure Fuel SIMPLE LIPIDS

They are esters of fatty acids with various alcohols. If the alcohol is glycerol, then they are called fats or neutral fats and are also called triglycerides as all the three hydroxyl groups of the glycerol are esterified. If the fat is liquid at ordinary temperature it is called an oil. Triglycerides are given by the formula

R = Same or different All of the three fatty acids can be same or different. If all the three fatty acids are same, then they are called simple triglycerides. If the fatty acids are different, then they are called mixed triglycerides. In nature, mixed triglycerides are more abundant than the simple triglycerides.

CHEMISTRY OF LIPIDS 55

If the alcohol is high molecular weight instead of glycerol then they are called waxes. Comparison of simple and compound lipids is terms of their composition. Lipid

Components

Simple lipids 1. Triglycerides 2. Waxes Compound lipids

1. 2. 3. 4.

Glycerol + Fatty acids Alcohol + Fatty acids (Both long chain) Phospholipids Glycerol + Fatty acids + Phosphate Sphingomyelins Sphingosine + Fatty acid + Phosphate + Choline Cerebrosedes (glycolipids) Sphingosine + Fatty acid + Simple sugar(s) Gangliosides (glycolipids) Sphingosine + Fatty acid + 2-6 simple sugars one of which is sialic acid

Fatty Acids Fatty acids in nature as such are not very abundant but are present as ester. Fatty acids are represented by general formula R—COOH. A fatty acid is a long chain aliphatic carboxylic acid. General points about them. 1. They are monocarboxylic acids. 2. Number of carbon atoms are even, though odd number fatty acids exist but are very rare. 3. They may be saturated or may be unsaturated. If unsaturated they can be monounsaturated acid or poly-unsaturated acid. Mammals and plants contain both monosaturated and polyunsaturated fatty acids whereas all the fatty acids containing double bonds that are present in bacteria are monounsaturated. Plant and fish fats contain more polyunsaturated fatty acids than animal fats. The double bonds in a polyunsaturated fatty acid are neither adjacent nor conjugated since this would

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make the structure to easily oxidisable when exposed to environment oxygen. Rather the double bonds are three carbon apart; this provide somewhat greather protection against oxidations. Fats obtained from animals are generally saturated and those from plants are commonly polyunsaturated. However, these are some exceptions: coconut, palm oils are highly saturated. The most common among the saturated fatty acids are palmitic acid (C16), stearic acid (C18) and among the unsaturated fatty acid, oleic acid (C18). Unsaturated fatty acids have lower melting point than saturated fatty acids of same chain length. Fatty acids with odd number of carbon atoms occur in trace amounts in terrestrial and marine animals. Fatty acids with one to eight carbons are liquids at room temperature while those with more carbon atoms are solids. The most common fatty acids in neutral fats are: No. of atoms Butyric acid Caproic acid Lauric acid Palmitic acid Stearic acid Oleic acid

4 6 12 16 18 18

Formula CH3—(CH2)2—COOH CH3—(CH2)4—COOH CH3—(CH2)10—COOH CH3—(CH2)14—COOH CH3—(CH2)16—COOH CH2—(CH2)7—CH=CH —(CH2)7 —COOH

Fats as an Energy Source Fats/oils are tremendous source of energy and 40% of total calories are provided by fatty acids that come from triglycerides and phospholipids. Naturally occurring straight chain saturated fatty acid No. of Common name C atoms 2 3 4

Acetic acid Propionic acid Butyric acid

⎫ ⎬ ⎭

Type

Systematic name

Short chain

n-Ethanoic acid n-Propanoic acid n-Butanoic acid

Contd...

CHEMISTRY OF LIPIDS 57

Contd... 8 10 12 14 16 18 20

Caprylic acid Capric acid Lauric acid Myristic acid Palmitic acid Stearic acid Arachidic acid

⎫ ⎬ ⎭

Medium chain

⎫ ⎬ ⎭

Long chain

n-Octanoic acid n-Decanoic acid n-Dodecanoic acid n-Tetradecanoic acid n-Hexadecanoic acid n-Octadecanoic acid n-Eicosanoic acid

The presence of double bond in the molecule gives rise to geometric isomerism. All naturally occurring unsaturated long chain fatty acids are found in cis isomer. Most plant fats are liquid since they contain a large proportions of unsaturated fatty acids with melting points. Animal fats, on the other hand, contain a high proportion of palmitic and stearic acids, and are solid or semi-solid at room temperature. Milk fat is unusual in containing a high proportion of shorter chain (C4-C14) fatty acids. Essential Fatty Acids They are also called polyunsaturated fatty acids. They are not synthesized in the body and hence, have to be provided in the diet. Although linolenic acid and arachidonic acid are synthesized by the body from linoeic acid, but they are synthesized in insufficient quantity for our needs. The deficiency of essential fatty acids in humans gives rise to dry, scaly skin, hair loss, poor wound healing, failure of growth and increase in metabolic rate. These essential fatty acids requirement is about 1% of the caloric intake be in the form of essential fatty acids. Essential fatty acids are needed for proper cell membrane formation and for synthesis of prostaglandins prostacyclins, thromboxanes and leukotrienes. Essential fatty acids are:

1. 2. 3. 4.

Linoleic acid Linolenic acid Arachidonic acid Timnodonic acid

No. of carbon atoms

No. of double bonds

18 18 20 20

2 3 4 5

Position of double bonds from carboxyl end

Dietary source

9, 9, 5, 5,

Vegetable oils Vegetable oils Vegetable oils Fish oils

12 12, 15 8, 11, 14 8, 11, 14, 17

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Vegetable oils are oils and have many double bonds hence polyunsaturated appears on the label of must vegetable oils. Butter, on the other hand, is a fat and hence would be expected to have saturated fatty acids, i.e. no double bonds. Two of the essential fatty acids, linoleic and linolenic acids are not synthesized by the mammal but are synthesized by plants. As long as adequate amounts of linoleic acids are available mammals can synthesize other essential acids.

Structures Linoleic acid CH 3(CH2)4CH=CHCH2CH=CH=(CH2)7COOH Linolenic acid CH3CH2CH=CHCH2CH=CHCH2CH =CH(CH2) 7COOH Arachidonic acid CH3(CH2)4(CH=CHCH2)4(CH2)2COOH Essential fatty acids are necessary in the biosynthesis of prostaglandins and for proper cell membrane formation. Prostaglandins are hormone-like compounds which in small amounts have profound effect. Important fatty acids in mammalian tissues: Common name

No. of carbon atoms

Double bonds

Acetic acid Lauric acid Myristic acid Palmitic acid Stearic acid Oleic acid Linoleic acid Linolenic acid Arachidonic acid

2 12 14 16 18 18 18 18 20

0 0 0 1 0 1 2 3 4

Position of double bonds

9 9 9, 12 9, 12, 15 5, 8, 11, 14

Prostaglandins Prostaglandins are the derivatives of prostanoic acid which are the cyclic derivatives of unsaturated fatty acids having twenty carbon atoms.

CHEMISTRY OF LIPIDS 59

Prostaglandins are synthesized from essential fatty acids such as linoleic acid, linolenic acid and arachidonic acid. Five type of rings are found in the naturally occurring prostaglandins giving rise to prostaglandins of A, B, E, F and G or H series. The prostaglandins which are widely distributed in the body are PGE1, PGE2, PGE3, PGF1α, PGF2α and PGF3α. Linolenic acid is the precursor to PGE3 and PGF1α, Arachidonic acid is the precursor to PGF2 and PGF2α.

Prostaglandins are synthesized and released by all mammalian cells and tissues except RBC. Also prostaglandins are not stored in cells but are synthesized and released immediately. Biological function of prostaglandins: 1. They lower blood pressure (PGE, PGA, PGI2). 2. They are used in the induction of labor, termination of pregnancy and prevention of conception (PGE2). 3. They are used in treatment of gastric ulcer (PGE). 4. They are used to prevent inflammation. 5. They are used in asthma. 6. They are used in congenital heart disease. 7. They inhibit platelet aggregation (PGI2) whereas PGE2 promote clotting process. Eicosanoids: Fatty Acid Derivatives Eicosanoids are all derived from C-20 carbon arachidonic acid. Prostaglandins, Thromboxanes and Leukotrienes are collectively referred as eicosanoids. They have a variety of extreme potent hormone like action on various tissues. These compounds are involved in the regulation of blood pressure, diuresis, blood platelet aggregation, effects on immune and nervous systems, gastric acid secretion and muscle contraction.

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Properties of Fats 1. Acrolein formation: When glycerol is heated in the presence of a dehydrating agent such as potassium bisulphate, acrolein is produced.

Acrolein has a characteristic unpleasant odor and is easily identified on the basis of this smell. This reaction occurs whether glycerol is in free or esterified form as occurs in the triglycerides. 2. Hydrogenation: Unsaturated fats can be hydrogenated by the addition of hydrogen across the double bonds of the fatty acids in the presence of nickel as catalyst to give fully saturated fats. The above process is called Hardening of oils whereby vegetable oils are hydrogenated to produce commercial cooking fats. 3. Saponification: Hydrolysis of a fat by alkali is called Saponification. The products of hydrolysis are glycerol and alkali salts of fatty acids, which are called soaps. Since the common fats contain palmitic acid, stearic acid and oleic acid predominantly, the soaps used for washing consist largely of sodium salts of these acids. While these fatty acids are insoluble in water their sodium and potassium salts are water soluble.

4. Rancidity: Rancidity is a chemical change resulting in unpleasant odor and taste on storage when fats are exposed to light, heat, air and moisture. Rancidity is more rapid at high temp-

CHEMISTRY OF LIPIDS 61

erature. Rancidity may be due to hydrolytic or oxidative change taking place at the double bonds of the unsaturated fatty acids resulting in short chain aldehydes or ketones which have unpleasant odor. The addition of certain substances, called antioxidants such as ascorbic acid and vitamin E prevents rancidity whereas addition of proxidants like copper, lead and nickel quickens rancidity. The oxidation of unsaturated bonds in fatty acids when the are exposed to oxygen in the environment is referred to as either auto oxidation or peroxidation. Rancid fats are those that contain an appreciable amount of peroxidized fatty acid. Antioxidants are generally added to many food fats to improve their storage quantities.

Characterization of Fats Saponification number: Saponification number is defined as the “milligrams of KOH required to saponify 1 gm of fat”. Since fats are mixtures of triglycerides largely of mixed type so the saponification number of a fat indicates the average molecular weight (average chain length) of the fatty acids constituting or comprising the fat. Saponification number is inversely proportional to the average chain length of the fatty acids. Higher the saponification number, the shorter will be the chain lengths of the fatty acids and vice versa. The saponification number of some of the fats is given below: Fat Butter fat Human fat Olive oil Cottonseed oil Linseed oil Coconut oil Castor oil

Saponification number 210-230 195-200 185-195 194-196 188-195 250-260 175-185

Iodine number: Iodine number of a fat is defined as the number of gm of iodine absorbed by 100 gm of the fat. Halogens, e.g. iodine or bromine are taken up by the fats because of the presence of double bonds present in the fatty acid part of the fat.

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Iodine number is a measure of the degree of unsaturation of fat. Iodine number of some of the fats is given below: Fat Butter fat Human fat Olive oil Peanut oil Corn oil Soyabean oil Linseed oil

Iodine number 26 - 28 65 - 70 80 - 90 85 - 100 105 - 115 135 - 145 170 - 200

Acid number: Acid number is defined as the milligrams of KOH required to neutralize the free fatty acids present in 1 gm. of fat. This is used in determining the rancidity due to free fatty acids. Acetyl number: The acetyl number is defined as the milligrams of KOH required to neutralize acetic acid liberated by the saponification of 1 gm of fat after it has been acetylated. Since acetylation takes place at the hydroxy groups of the hydroxy fatty acid residues in the fat, so acetyl number is a measure of the hydroxy fatty acids in the fat content. Polenske number: The ml of N/10 KOH required to neutralize the insoluble fatty acids from 5 gm. of fat which are not steam volatile. Reichert Meissel number: This represents the ml of N/10 KOH required to neutralize the volatile acid obtained from 5 gm of fat which has been saponified then acidified to liberate the fatty acids and then steam distilled. Butter fat, which contains shorter chain fatty acids has a Reichert Meissel number of 26 to 30. COMPOUND LIPIDS They are the esters of fatty acids containing nitrogen base in addition to an alcohol and fatty acids.

CHEMISTRY OF LIPIDS 63

A molecule which has changed and an unchanged portion is called an amphipathic molecule. Phospholipids They are also known as phosphatides. Phospholipids act as a detergent and increase the solubility of other lipids. They are present in all cells as well as in the plasma. Phospholipids include the following groups:

Phosphatidic Acid The general structure of phosphatidic acid. They are important intermediates in triglyceride synthesis. Phosphatidic acid on hydrolysis yield glycerol, fatty acid and phosphoric acid.

Lecithins The structure of lecithins are:

Lecithin contains saturated fatty acid residue at the α-position and unsaturated fatty acid residue at the β-position of the glycerol. Lecithins on hydrolysis give glycerol, fatty acid, phosphoric acid and choline.

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Cephalins The structure of cephalins are:

Cephalins differ from lecithins with respect to base attached to phosphoric acid. If the base is ethanol amine then it is called phosphatidyl ethanolamine or ethanolamine cephalin. If the base is amino acid serine then it is called phosphatidyl serine which is also called serine cephalin. Cephalins on hydrolysis yield glycerol, fatty acids, phosphoric acid, ethanol amine or serine.

Phosphatidyl Inositol The structure of phosphatidyl inositol is: It contains inositol in place of base.

CHEMISTRY OF LIPIDS 65

Cardiolipin An important phospholipid of mitochondrial membrane is cardiolipin. It is a diphosphatidyl glycerol in which two phosphatidic acids are joined by a molecule of glycerol. These phospholipids are particularly rich in the polyunsaturated fatty acids especially linoleic acid.

Plasmalogens These compounds possess fatty aldehyde in place of fatty acid at the α-position, with the result the normal ester linkage is replaced by the ether linkage on the C1 carbon. In some cases, bases like choline, serine or ethanol amine are also found. They are found in brain and heart.

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Sphingomyelins Phospholipids containing sphingosine are called sphingomyelins. They contain, a complex base sphingosine in addition to phosphoryl choline. A fatty acid is attached to the amino group of the sphingosine. No glycerol is present.

Structure of Sphingomyelins

Sphingomyelins are present in all tissues especially in brain and other nervous tissues. Sphingomyelins on hydrolysis yield sphingosine, fatty acid, phosphoric acid and choline. Increased concentration of sphingomyelins occur in liver, spleen, etc. in a condition known as Niemann-Picks disease. Cerebrosides or Glycolipids Glycolipids are carbohydrate-glyceride derivatives containing sugar, sphingosine and a fatty acid. These compounds do not contain phosphoric acid. If the sugar component is galactose, the lipid is termed galactolipid. The term cerebroside is used because it is found in large quantities in brain tissues particularly in white matter.

CHEMISTRY OF LIPIDS 67

Cerebrosides

On hydrolysis cerebrosides give sphingosine, a fatty acid and galactose. Cerebrosides are differentiated on the basis of fatty acid present. Examples: Kerasin: It contains Lignoceric acid Cerebron: It contains Hydroxy Lignoceric acid Nervon: It contains Nervonic acid Oxynervon: It contains Hydroxy Nervonic acid Cerebrosides occur in large amounts in the white matter of brain and in the myelin sheaths of nerves. In Gaucher’s disease, large amount of cerebroside accumulates in the liver and spleen. Gangliosides They are found in nerve tissues. They contain carbohydrates, N-acetyl galactosamine and N-acetyl neuraminic acid.

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Sulfatides (Sulpholipids) They are cerebrosides having a sulfate group attached to the galactosyl residue. DERIVED LIPID Those substances which are derived from the above two groups by hydrolysis. These include fatty acids of various series, steroids, bile acids and substances associated with lipids in nature such as carotenes, vitamin A, D, E and K. Lecithins are hydrolyzed by certain enzymes, phospholipases or lecithinases. The nature of hydrolysis depends upon the type of phospholipase used. Phospholipase A: Present in snake venom (cobra) hydrolyzes fatty acid in α or 1-position of glycerol in the lecithin to form lysolecithins. In the similar manner it acts on cephalin. Phospholipase B: Hydrolyzes the remaining fatty acid of lysolecithin present at β or 2-position to form glyceryl phosphorylcholine. Phospholipase C: Hydrolyzes phosphorylcholine from lecithins to form diglycerides. Phospholipase C catalyses the hydrolysis at the glycerol side of the phosphate group. Phospholipase D catalyses the hydrolysis on the phosphate side of the phosphate group.

CHEMISTRY OF LIPIDS 69

Phospholipase D: Hydrolyzes choline from phosphatidyl ethanolamine (cephalin) form phosphatidyl serines. There are two classes of nonsaponificable lipids. Terpenes They are linear or cyclic compounds formed by condensation of two or more isoprene units.

Other important terpenoid compounds are: a. Tocopherol (vitamin E) b. Coenzyme Q (also called ubiquinone) c. Vitamin K (a naphthaquinone) They include vitamins A, E, K and carotenes, etc.

Cyclopentano-perhydro-phenanthrene (Steroid nucleus)

ring

Steroids The term steroids includes many compounds which have however one feature in common, the steroid skeleton. Steroids are the derivatives of cyclopentano-perhydro-phenanthrene ring (consists of four fused rings). This is a saturated (perhydro) pheranthrene ring with a cyclopentane ring attached. Steroids are steroidal alcohol. The most important member of this group is cholesterol. The four rings that make up

70 BIOCHEMISTRY FOR STUDENTS

perhydro-cyclopentano-phenanthrene are named alphabetically from left to right. Despite popular belief, cholesterol is not a poison but a very necessary part of our cell membranes and the basis of sexual hormones (androgens, estrogens, etc). Cholesterol is only a problem if it is in excess and in this respect we do not need cholesterol in our diets because body can synthesis it. Steroids belong to the class of important biological compounds with diverse physiological activities. Some of the biologically important steroids are: a. Ergosterol: b. Bile acids: c. Adrenal cortex steroids: d. Female hormones: e. Male sex hormones:

UV radiation causes rupture of ring B to produce vitamin D. In lipid metabolism. Corticosterone and cortisol. Progesterone and estrogen. Testosterone and androsterone.

Cholesterol is an animal fat and it does not occur in plants. Cholesterol contains hydrogen group at C-3, methyl groups at C-10 and C-13, a double bond at C-5 and an 8C branched alkyl group attached to C-17. This marks a total of 27C. This ring structures are lipid soluble and hydroxyl group of C-3 is hydrophilic.

CHEMISTRY OF LIPIDS 71

Plants have stigmasterol and β-sitosterol which differ only in the alkyl group side chain attached at C-17. The Antioxidant System In healthy individuals, the antioxidant system defends tissues against free radical attack. Antioxidants are known to prevent cellular damage and enhance repair. Three classes of antioxidants have been identified. a. Primary antioxidants: They prevent the formation of new free radical species, e.g. superoxide dimutase, glutathione peroxidase, ceruloplasmin, transferrin, ferritin. b. Secondary antioxidants: They remove newly formed free radicals before they can initiate chain reactions. These chain reactions can lead to cell damage and further free radical formations, e.g. vitamin E, vitamin C, β-carotene, uric acid, bilirubin, albumin. c. Tertiary antioxidants: They repair cell structures damaged by free radicals attack, e.g. DNA repair enzymes, methionine sulphoxide reductase. Deficiency in the antioxidant system can develop for a number of reasons: a. Low intake of dietary antioxidants b. Total parenteral nutrition c. Decreases that reduce the absorption of antioxidant nutrients from food, e.g. Crohn’s disease d. Renal dialysis In these situations the antioxidant system struggles to protect the body from free radical attack and as a result the risk of free radical-mediated disease increases. Increased antioxidant status by supplementation may indeed reduce the risk of certain diseases. i. High intake of vitamin E has been associated with reduced risk of mortality from ischemic heart disease. ii. High incidence of vitamin C and β-carotene have been associated with a reduced incidence of some cancers. iii. Dietary supplementation of vitamin E, β-carotene and selenium significantly reduces mortality from esophageal cancer. iv. Within one week on antioxidant rich, low fat diet reduces lipid peroxide levels and increased aborrhic acid level in patient in the acute myocardial infarction.

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Free Radicals A free radicals is defined as any atom or molecule that possesses an unpaired elactron. It can be anionic, cationic, or neutral. Free radicals are highly reactive molecules generated by the biochemical redox reactions that occur as part of normal cell metabolism and by exposure to environmental factors such as UV light, cigarette smoking, environmental pollutions and gamma radiations. Human body is constantly under attack from free radicals. Some toxic compounds can result in the production of free radicals which include anticancer drugs, anaesthetics, analgesics, etc. The free radicals species which occur in the human body are: a. Superoxide radical (•O2¯) b. Hydroxyl radical (OH•) c. Nitric oxide radical (NO•) d. Peroxyl radical (ROO•). Once formed, free radicals attack cell structures within the body. As a result, free radicals have been implicated in numerous diseases such as atherosclerosis, cancer, AIDS, liver damage, rheumatoid arthritis, Parkinson’s disease, etc. Process of Lipid Peroxidation This process is responsible for randicity of food. This process involves: i. Initiation ii. Propagation iii. Termination.

Initiation +

ROOH + Metaln+ → ROO• + Metal(n-1) + H+ X• + RH → R• + HX

Propagation R + O2 → ROO• ROO• + RH → ROOH + R•

CHEMISTRY OF LIPIDS 73

Termination 2ROO• → ROOR + O2 ROO• + R•→ ROOR R• + R• → RR Eicosanoids Eicosanoids are formed from C20 polyunsaturated fatty acid. Arachidonate and some other C20 fatty acids give rise to eicosanoids which includes prostaglandins, thromboxanes, leukotrienes, lipoxins. There are two pathways of their formation: 1. Cyclooxygenase pathway 2. Lipooxygenase pathway.

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CHAPTER

4

Chemistry of Amino Acids and Proteins CHEMISTRY OF AMINO ACIDS

Naturally occurring amino acids are amino acids containing amino group and carboxyl group on the same alpha carbon atom and are represented by the general formula:

All amino acids found in living systems, plant and animal proteins are L-α-amino acids. Glycine is the only amino acid, which is optically inactive and cannot be resolved into D-or L-form because of symmetry on the α-carbon atom. All other amino acids are optically active. The configuration of L-α-amino acid is:

A variety of classification of amino acids are possible. Either they can be classified according to the presence of acidic, basic or neutral groups or upon their chemical structures, i.e., presence of polar groups, nonpolar groups, sulphur containing groups, aromatic groups, heterocyclic ring, branched chain and so on.

CHEMISTRY OF AMINO ACIDS AND PROTEINS 75

Classification of Amino Acids 1. Aliphatic amino acids 2. Aromatic amino acids 3. Heterocyclic amino acids.

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CHEMISTRY OF AMINO ACIDS AND PROTEINS 77

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Tryptophan

Besides these there are number of amino acids which are obtained in free or combined form but do not occur in protein molecules, e.g. thyroxine, triiodothyronine, ornithine, citruline, α-aminobutyric acid, β-alanine, etc. Their structures are given here.

CHEMISTRY OF AMINO ACIDS AND PROTEINS 79

A dipeptide has two amino acids joined by a single peptide bond; a tripeptide is composed of three amino acids joined by two peptide bonds: a polypeptide is one in which any number (n) of amino acids or (AA)n are linked together by (n-1) peptide bonds. Examples of relatively smaller peptides that possess biological activity are glutathione, oxytocin, vasopressin, hypertensin, etc. Glutathione is a tripeptide consisting of glutamic acid, cystine and glycine and is found in red blood cells.

Oxytocin and vasopressin are produced by the posterior of the pituitary gland. Each is made up of nine amino acids. Oxytocin causes contraction of smooth muscle and it is used in obstetrics to initiate labor whereas vasopressin raises blood pressure and reduces the secretion of urine. Angiotensin I has 10 amino acids and angiotensin II has 8 amino acids. They cause hypertension. Functions of Amino Acids Amino acids serve as: 1. Building block of proteins 2. Precursors of: a. Hormones. (peptide and thyroid) b. Purines c. Pyrimidines d. Porphyrins e. Vitamins 3. Neurotransmitter such as tryptophan (sertonin). 4. Transport of nitrogen: Alanine, glutamine. 5.Substrates for protein synthesis: Those for which there is a codon.

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Essential Amino Acids Those amino acids which are not synthesized in the body and hence have to be provided in the diet. They are also called indispensible amino acids. There are eight essential amino acids. They are leucine, isoleucine, threonine, tryptophan, phenylalanine, valine, methionine and lysine. Adequate amounts of essential amino acids are required to maintain the proper nitrogen balance. Deficiency of one or more essential amino acids in the diet gives rise to decrease in protein synthesis resulting in failure in growth of the child, negative nitrogen balance in adults and fall in plasma proteins and hemoglobin levels. Semiessential Amino Acids Those amino acids which are synthesized partially by the body but not at a rate to meet the requirement of the body are called semiessential amino acids. Semiessential amino acids are arginine and histidine.

Nonessential Amino Acids Those amino acids which are synthesized by the body. These amino acids are derived from carbon skeletons of lipids and carbohydrates during their metabolism or from the transformation of essential amino acids. Nonessential amino acids are alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine and tyrosine. Nitrogen Balance The ratio of: Intake N = 1, i.e. nitrogen equilibrium. Normal adults Output N are in nitrogen equilibrium. > 1, i.e. positive nitrogen balance, e.g. during pregnancy, convulsions and growth. < 1, i.e. negative nitrogen balance, e.g. in malnutrituion and in certain wasting diseases where, there is tissue breakdown.

CHEMISTRY OF AMINO ACIDS AND PROTEINS 81

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Ninhydrin Reaction All amino acids (except proline and hydroxyproline), proteins or protein derivatives containing free amino group and a free carboxyl group react with ninhydrin to give a blue-violet colored compound called Rheumann’s purple, whereas amino acids, proline and hydroxyproline, give a yellow color with ninhydrin. Reaction with Nitrous Acid α-amino acids are deaminated to the corresponding α-hydroxy acids with nitrous acid. Each amino group yields one molecule of nitrogen which can be measured accurately. Hence, this reaction is used for the estimation of free amino groups in amino acids, peptides and proteins.

Formal Titration Sorensen’s formal titration method is used for the estimation of free carboxyl group in amino acid and mixtures of amino acids. By this method one can determine the rate of digestion of proteins by determining the increase in carboxyl groups which accompanies during enzymatic hydrolysis. Amino acids by virtue of Zwitter ion formation are neutral in solution. If formaldehyde is added to a solution of amino acid, an adduct is formed at the amino group, leaving the carboxyl group free and the molecule acidic in reaction. In other words the presence of formaldehyde decreases the basicity of the amino group, permitting free carboxyl group to exert its maximum acidity. Free carboxyl group thus can be titrated.

CHEMISTRY OF AMINO ACIDS AND PROTEINS 83

Isoelectric Point of Amino Acids (pl) pl is defined as that pH at which the amino acid does not migrate in an electric field. At this pH, the amino acid molecule exists in the Zwitter ion form, in which the sum of the positive charges are equal to the sum of the negative charges and the net charge on the molecule is zero. pl is calculated as:

where pK1 is the pH at which the carboxyl group is halftitrated and pK2 is the pH at which the N+H3 group is halftitrated. Amino acids are amphoteric electrolytes, i.e. they exhibit properties of both an acid and a base. The acidic groups of amino acids are carboxylic group (–COOH → –COO¯+ H+) and protonated α-amino group (–N+H3 → NH2 + H+). Basic groups of amino acids are dissociated carboxyl group (–COO¯ + H+ → –COOH) and α-amino group (–NH2 + H+ → –N+H3). Amino acids in aqueous solutions have been shown to occur as a dipolar species or zwitter ion (Molecules which have both a negative and a positive change). As every amino acid has at least two ionizable groups, it can exist in different ionic forms depending on the pH of the medium. In aqueous solution a neutral amino acid is in the zwitter ion form which is dipolar. It is therefore an amphoteric electrolyte. Ampholytes are those molecules that act as both an acid and a base.

In strongly acid pH, it is in cationic form while in strongly alkaline pH, it is in anionic form. At isoelectric pH, the solubility and buffering capacity is minimum.

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Similarly, protons exist as cations in the acid media and anion in the alkaline media of the isoelectric pH. Hence, protein acts as buffers on both sides of isoelectric pH. So a proton is an anion at pH values above the pl and is a cation at pH values below the pl. At the isoelectric pH, glycine exists as Zwitter ion. Addition of acid converts it into cation and addition of alkali converts it into anion. Therefore amino acids depending on the medium pH carry net zero, positive or negative charges. Q.

Show the formula of isoelectric glycine. Indicate by formulae what happens on the addition of (a) acid and (b) base to the isoelectric molecule. Ans. At isoelectric point the glycine exists as:

CHEMISTRY OF AMINO ACIDS AND PROTEINS 85

PROTEINS Proteins are defined as compounds of high molecular weight made up of α-amino acids linked to one another by peptide linkages. Proteins contain 20 odd individual amino acids present in characteristic proportions and linked in a specific sequence in each protein. Proteins are linear polymers consisting of L-α-amino acids. The amino acids are joined together by peptide bonds. The peptide bond is formed by the union of carboxyl group of one amino acids with amino group of other amino acid with an elimination of water molecule. Classification of Proteins Proteins are classified on the basis of their composition.

Simple Proteins Simple proteins are made up of amino acids only and on hydrolysis yield constituent amino acid’s mixture only. Example: 1. Fibrous proteins: These are animal proteins which are highly resistant to digestion by proteolytic enzymes. They are water insoluble. a. Collagens It contains high proportion of hydroxy proline and hydroxylysine. It is a major protein of connective tissues. On boiling with water it forms gelatin. b. Elastins It is present in tendons and arteries. c. Keratins It contains large amount of sulphur as cystine. It is present in hair, wool, nails, etc. 2. Globular proteins: a. Albumins Serum albumin and ovalbumin of egg white. It is water soluble. It is precipitated from solution by full saturation of ammonium sulfate. It is coagulated by heat.

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b. Globulins

c. Glutelins

d. Gliadins (Prolamines) e. Protamines f. Histones

Serum globulins, fibrinogens and muscle myosin. It is soluble in dilute salt solutions. It is precipitated from solution by half saturation of ammonium sulphate. It is coagulated by heat. Cereal proteins such as glutelins of wheat, oxyzenin from rice and zein of maize. It is soluble in weak acids or bases but insoluble in neutral aqueous solutions. Gliadin from wheat and zein from corn. It is water insoluble but soluble in ethanol. Salmine from salmon sperm cells contains high proportion of arginine. Globulin in hemoglobin. It contains high proportion of basic amino acid. It is water soluble.

Conjugated Proteins They are proteins which contain nonprotein group (also called prosthetic group) attached to the protein part. On hydrolysis they give nonprotein component and amino acid mixture. Conjugated Protein = Protein part + Prosthetic group. Conjugated proteins are classified according to the nature of the nonprotein group attached to the protein part.

Derived Proteins They are formed from simple and conjugated proteins by physical and chemical means. The products of partial hydrolysis of proteins are often classified as derived proteins. 1. Primary derived protein a. Protein

These are formed as a result of slight change in structure with little or no hydrolytic cleavage of peptide bonds. Fibrin from fibrinogen.

CHEMISTRY OF AMINO ACIDS AND PROTEINS 87 Proteins

Prosthetic group

1. Nucleoproteins 2. Phosphoproteins

= =

Nucleic acid Phosphoric acid

3. Glycoproteins

=

4. Lipoproteins

=

5. Flavoproteins

=

Carbohydrate or a derivative of carbohydrate Lipids (Lecithin, Cephalin, cholesterol, etc). Riboflavin

6. Metalloproteins

=

b. Metaprotein c. Conjugated proteins 2. Secondary derived protein

a. Proteoses b. Peptones c. Peptides.

Metals (Zinc, iron and copper)

Example Virus proteins Casein of milk (Serine residues are phosphorylated), ovovitellin of egg yolk. Mucin of saliva Serum lipoproteins Biological oxidation reduction reactions Carbonic anhydrases, catalase, cyctochrome oxidase

They are soluble in dilute acids and bases but insoluble in neutral solvents. Formed by the action of heat, alcohol, UV light, X-rays. Example, cooked egg white and egg albumin. These are formed by the progressive hydrolytic cleavage of the peptide bonds of protein molecules. They are water soluble and are not coagulated by heat.

These proteins are formed as a result of various deep seated changes in the structure or composition of the proteins. Separations of proteins by ion exchange resins in a chromatography is also an important technique for the separation and characterization of proteins by change. Ion exchange resins

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are prepared of insoluble materials such as agarose, polyacrylamide, cellulose, etc. that contains negatively changed ligands (such as –CH2COO¯, –C3H6SO3¯) or positively charged legands such as diethyl amino. The degree of retardation of a protein or amino acid by a resin will depend on the magnitude of the charge on the protein at a particular pH of the experiment. Molecule of the same charge as the resin are eluded first in a single band, followed by proteins with an opposite charge to that of the resin. Electrophoresis If a solution of a mixture of proteins is placed between two electrodes, the charged particle will migrate to one electrode or the other at a rate that depends on the net change and, depending on the supporting medium used, on the molecular weight. Structure of Proteins Proteins exhibit four levels of organization: Primary structure Secondary structure Tertiary structure Quaternary structure

Refers to amino acid sequence. Refers to folding of polypeptide chain into specific coiled structure which is repititive in one direction. Refers to arrangement and interrelationship of twisted chain into a three dimensional structure. Refers to the association of different monomeric subunit into a composite polymeric protein.

Primary Structure It determines the sequence of amino acids in the protein molecule. It indicates the number of amino acids, type of amino acids and in which fashion they are linked up. The sequence of amino acids in proteins can be found out by Sanger’s and Edman’s degradation method.

CHEMISTRY OF AMINO ACIDS AND PROTEINS 89

Sanger’s Method This method is used to determine the N-terminal amino acid of proteins. The reagent used is 2, 4-dinitrofluorobenzene (DNFB). DNFB reacts with free amino group of the terminal amino acid of proteins to give a yellow colored 2, 4-dinitrofluorobenzene derivative which on hydrolysis, give the terminal amino acid as the yellow 2,4-dinitro derivative and all the other amino acids of protein are obtained as free amino acids. The yellow derivative is separated and identified by paper chromatography, by comparison with known 2,4-DNP amino acid.

Edman’s Method N-terminal amino acid residue of proteins can also be identified by Edman’s method. The reagent used is Phenylisothiocyanate (PITC). It reacts with free alpha amino group of the N-terminal amino acid of proteins to give the phenylisothiocarbamate derivative of the protein which cyclizes in acid medium giving N-terminal amino acid as phenylthiocarbamyl amino acid (PTCA), leaving the rest of the protein chain intact, but shorter

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by one amino acid. PTCA then cyclizes to give the corresponding phenylthiohydration derivative, which is separated and identified by chromatography. The reaction with phenylisothiocyanate is then repeated on the shortened peptide. The amino acid sequence is thus determined from the N-terminal end of the peptide one by one.

Phenylthiohydantoin Derivative Edman’s method is superior over Sanger’s method. Edman’s degradation involves the removal of one amino acid at a time from the amino end of a peptide or protein chain, leaving the remaining peptide chain intact. The process can be repeated and the sequence of amino acid from N-terminal end is obtained. Whereas in Sanger’s method, only the N-terminal amino acid is identified because after the removal of N-terminal acid with DNFB, the remaining peptide chain breaks into amino acid mixture. Another reagent often used is Dansyl chloride (Dimethyl aminonaphthalene-5-sulphonyl chloride).

CHEMISTRY OF AMINO ACIDS AND PROTEINS 91

The procedure with this reagent is the same as that used with DNFB. A covalent bond is formed with the free N-terminal amino group. The dansylated protein is hydrolyzed with acid and dansylated amino acid is separated and identified by chromatography. C-terminal residues are usually identified with enzyme carboxypeptidase. This enzyme attack only the peptide bond joining the last residue with a free α-carbonyl group of the peptide chain. Amino acids released are identified by chromatography. Also the polypeptide is treated with the anhydrous hydrazine, which breaks peptide bonds forming hydrazides with the carbonyl carbons. The C-terminal residue does not form a hydrazide because its carboxyl group is free. After the removal of the hydrazides, this amino acid is then identified chromatographically.

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The repetition of Edman reactions under favorable conditions can be carried out for 30 to 40 amino acids into the polypeptide chain from the NH2-terminal end. Since most polypeptide chains in proteins contain more than 30 to 40 amino acids, they have to be hydrolyzed into smaller fragments and sequenced in sections. Both enzymatic and chemical methods are used to break polypeptide chains into smaller polypeptide fragments. Trypsin and chymotrypsin are proteolytic enzymes that are used for partial hydrolysis of polypeptide chains in sequencing. Enzyme trypsin catalyse the hydrolysis of peptide bond on the α-COOH side of the basic amino acid residues of lysine and arginine with the polypeptide chains. Chymotrypsin hydrolyzes peptide bonds on the α-COOH side of amino acid residues with larger apolar side chains. The chemical reagent cyanogen bromide cleaves peptide bonds on the carboxyl side of methionine residue with polypeptide chains.

R1 Phenylalanine Tyrosine Tryptophan Anginine, Lysine Methionine Tryptophan

Reagent Chymotrypsin Trypsin Cyanogen bromide O-Iodosobenzoic acid

Secondary Structure The polypeptide back-bone does not assume a random threedimensional structure, but instead generally forms regular arrangements of amino acids that are located near to each other in linear sequence. These arrangements are called as secondary structure of proteins. The durameter of helix is 10Å. This is of following types: 1. α-helix: This is most common type of secondary structure, it is spiral structure. α-helix is stabilized by extensive hydrogen bonding and it consists of 3-6 amino acid per turn. Proline disrupts the α-helical structure because it

CHEMISTRY OF AMINO ACIDS AND PROTEINS 93

is imino acid and geometricallly not compatible with helical structure. 2. β-sheet: In this surface appears pleated. So also known as α-pleated sheet. The two or more chains may be parallel or antiparallel. Amyloid protein deposited in brains of individuals with Alzheimer’s disease is composed of β-pleated sheet. 3. β-bends: β-bends reverse the direction of a polypeptide chain, helping it to form a compact, globular shape. These are usually found on the surface of protein molecules.

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4.Nonrepetitive secondary structure: About half of an average globular protein is organized into repetitive structures. These are not random but have a less regular structure. 5.Supersecondary structures: These mainly form core, i.e. interior to molecule. These are also known as motifs. The common ones are β-α-β unit, greek key and β meander.

Tertiary Structure Tertiary structure refers to the coiling of several helical portion of single helix into a three-dimensional structure. The tertiary structure of proteins is stabilized by: 1. Hydrogen bonding: It is formed by sharing of hydrogen atom between electronegative oxygen atoms, nitrogen atoms or combination of two. 2. Disulphide bonding: This results from electrostatic attraction between positively and negatively charged spacies. 3. Ionic interactions or salt bridges: These are nonpolar bonds between hydrocarbon containing compounds. 4. Ester bonding. 5. Hydrophobic interactions: These are the result of mutual interaction of electron and nuclei of molecules. 6. van der Waal’s forces.

Quaternary Structure Proteins containing more than one polypeptide chain display fourth level of structural organization called quaternary structure. In quaternary structure of proteins, the individual polypeptide chains are arranged in relation to each other so as to give a single three dimensional structure of the overall protein molecule. Each polypeptide chain in such a protein is called a subunit. Depending upon the number of subunits such proteins are called dimers, tetramers or polymers, etc. The various examples are hemoglobin, ferritin, etc. Reactions of Proteins 1. They give biuret test positive. 2. They give blue color with ninhydrin.

CHEMISTRY OF AMINO ACIDS AND PROTEINS 95

Biuret Reaction The name of the reaction is derived from the organic compound, a biuret, obtained by heating urea at high temperature which gives this test positive. The compound biuret contains two peptide linkage.

Biuret test is given by those compounds which contain two or more peptide bonds. Since proteins are polypeptides hence, it is a general test for proteins. When proteins are treated with alkali and minute quantities of cupric ions, a pink or purple color is obtained.

Precipitation Reactions Proteins are precipitated from the solution by a large number of reagents and the process is called deproteinization. Such precipitation reactions are important in the isolation of proteins, in the deproteinization of blood and other biological fluids. a. Effect of salt concentration: Proteins are precipitated from the solution by the addition of (NH4)2SO4 and Na2SO4. Addition of large amounts of ionic salts results in increase in protein: protein interaction and decrease in protein: water interaction, the process is called salting out.

96 BIOCHEMISTRY FOR STUDENTS

b. Effect of positive ions: The positive ions most commonly used for protein preci- pitations are heavy metal cations such as Cu++, Zn++, Fe+++ etc. These cations precipitate proteins from alkaline solution by combining with the negatively charged protein to form an insoluble precipitate of metal proteinate. c. Effect of negative ions: Addition of tungstic acid, phosphotungstic acid, trichloroacetic acid, picric acid, sulphosalicylic acid results in precipitation of protein in acidic solution. Denaturation Denaturation is the unfolding of the characteristic native folded structure of the polypeptide chain of protein. Comparatively weak forces responsible for maintaining the secondary, tertiary and quaternary structure of proteins are rapidly disrupted during the denaturation. The primary structure held by covalent peptide bonds however is not disrupted. After denaturation such proteins acquire the random coil structure which may renaturate into native form under favorable conditions. Denaturation of oligomeric protein involves the (i) dissociation of subunits peptide chains from each other with or without (ii) the unfolding of individual chains into random coils. Such proteins usually are unable to renaturate or refold into the natural form. There are two conspicuous changes that often result from denaturation. 1. Loss of (Partial or Complete) biological activity of the protein. 2. The solubility decrease drastically, i.e. almost the precipitation takes place. Denaturation results in loss of biological activity caused by heat, pH changes, by organic solvents, effect of radiation, etc. In electrophoresis, an ampholyte such as protein, peptide or amino acid in a solution buffered at a particular pH is placed in an electric field. Depending on the relationship of the buffer pH to the pI of the molecule, the molecule will either move toward the cathode (–) or the anode (+) or remain stationary (pH = pI).

CHEMISTRY OF AMINO ACIDS AND PROTEINS 97

For plasma protein separation the solution is buffered at pH 8.6 which is at a pH substantially above the pI of the major plasma proteins. The proteins are negatively charged and move toward the positive pole. The peaks are obtained according to their rate of migration in order of their pI values are these of albumin, α1-, α2- and β-globulins, fibrinogen and α1- and α2-globulins. The different major proteins are designated underneath the peaks. The direction of migration is from right to left.

Electrophoretic pattern of normal serum

Functions of Proteins in the Body 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Catalytic proteins: Enzymes Structural proteins: Collagen Contractile proteins: Actin, myosin Natural defence proteins (Immunity): Antibodies Transport proteins: Albumin, Globulin, Hemoglobin, ceruloplasmin, apolipoprotein Blood proteins: Fibrinogen Hormonal proteins: Insulin Respiratory proteins: Cytochromes Repressor proteins: Regulate expression of genes of chromosomes Rece ptor proteins: Transport information to cell interior after interacting with proteins on the outside. Ribosomal proteins: Associated in the proteins synthesis Toxin proteins: Venoms

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13. Vision proteins: Rhodopsin 14. Storage: Ferritin. Plasma Proteins Normal value of plasma proteins is 6 to 8 gm per 100 ml of blood. Plasma protein include albumin, globulin and fibrinogen. They can be separated. 1. By precipitation method using sodium sulfate, ammonium sulfate, etc. 2. By electrophoresis. In normal human plasma, 6 fractions have been separated by electrophoresis. They are: i. Albumin ii. α1-globulin iii. α2-globulin iv. β1-globulin v. γ2-globulin vi. Fibrinogen. Functions of Plasma Proteins 1.

Osmotic pressure: Plasma proteins are important in regulating water between blood and tissues. Small molecules of plasma and tissue fluid such as glucose, amino acids, urea, electrolytes, freely diffuse back and forth and hence, exert the same osmotic pressure in both fluids, i.e. on both sides of capillary. However, plasma and lymph protein do not freely diffuse through the capillary walls and since the prot ein concentration of plasma is much higher than of lymph by difference in the protein osmotic pressure of the two fluids. This difference in the osmotic pressure of lymph and plasma is estimated to average about 22 mm Hg and represents effective osmotic pressure of plasma.

CHEMISTRY OF AMINO ACIDS AND PROTEINS 99

2. 3. 4.

5.

As buffers: Proteins are amphoteric in nature and thus help in maintaining pH of the body. Reserve proteins: Proteins serve as source of proteins for the tissues when the need arises. As carrier of certain metabolites: The transport of certain insoluble substances such as bilirubin, free fatty acids, steroid hormones and lipids is carried out by various fraction of serum proteins. As immunoglobulins: The property of antibodies formation resides in γ-globulin fraction of the proteins.

Immunoglobulin Immunoglobulins or antibodies, make up the γ-globulins fraction of the plasma. These defensive proteins are synthesized in response to exposure to a foreign material usually a protein or complex carbohydrate. The foreign material is called antigen. The formation of antibodies affords immunity against the antigen and this response is protective. Immunoglobulins are composed of four polypeptide chains, two light chains (L-chains) and two heavy chains (H-chains) per molecule. These chains are linked by disulphide bonds. There are two classes of light chains, κ and λ thus creating two series of immunoglobulin molecules. Each class of immunoglobulin contains a unique type of heavy chain. These are designated as ρ, α, μ, δ and ε chains. These immunoglobulins and their chemical formulae are represented as follows: Immunoglobulins IgG IgA IgM IgD IgE

H-chains

K-type

ρ α μ δ ε

K2r 2 K2α 2 K 2 μ2 K2δ2 K2ε2

λ-type λ2r2 λ2α2 λ2μ2 λ2δ2 λ2ε2

Immunoglobulins also called Antibodies, comprises the gamma-globulin fraction of the plasma. They are synthesized in the body in response to the exposure (or administration)

100 BIOCHEMISTRY FOR STUDENTS

to a foreign moiety called Antigen. The foreign material or antigens are usually proteins or carbohydrates. The formation of antibodies give rise to immunity against the antigen and this response is protective. The immunoglobulins are glycoproteins containings 3% to 12% carbohydrates including D-mannose, D-galactose, Lfucose, D-glucosamine and a sialic acid. On ultracentrifugation the immunoglobulins are separated into three major fractions IgM, IgG and IgA. Two other immunoglobulins IgD and IgE occur in plasma in small amount. Most of the antibodies are in IgG fraction which represents 70% of the total r-globulins. Immunoglobulins are made up of subunit peptide chains called heavy chain (mol wt 40,000) and a light chain (mol wt 20,000). The three types of heavy chain are μ, r and α. Two types of lighter chain are K and λ. Both types of light chains contain a segment with a constant sequence of amino acids comprising about half the chains and a variable portion of other half. Heavy chains also contain a variable portion of amino acid sequence (about 110 amino acids) and 330 amino acids forming the constant portion of the chains. The variable portion of the light and heavy chains of immunoglobulins contains the active sites of the molecule. Light chains and heavy chains are linked together in the whole immunoglobulin molecule by means of disulphide linkages. Type

Subunit composition

Mol wt

Carbohydrate content (%)

Serum level mg/100 ml

IgG

γ2k2, γ2λ2

153,00

3

0.81.6

IgA

(α2k2)n, n(α2γ2) n = 1 to 4

180,0005000,000

5-10

0.2-0.4

lgM

(μ2k2)n, (μ2γ2)n n = 5,6

900,000

10-10

0.2-0.5

CHEMISTRY OF AMINO ACIDS AND PROTEINS 101

Each heavy chain has four interchain disulfide bonds; two between the pair of μ-chains in the monomer one to the light chain, and one intersusunit bridge between the monomers. The light chains are denoted by smaller lines and may be of the κ or γ type. The solid circles attached to the heavy chains of one of the monomers represent complex oligosaccharides.

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CHAPTER

5

Hemoglobin

PORPHINS Porphins are cyclic compounds formed by the linking of four pyrrole rings through methane bridges (—CH=).

The four pyrrole rings are labeled as I, II, III and IV and the bridges as α, β, γ and δ. Substituents on the rings are labeled as 1, 2, 3, 4, 5, 6, 7, and 8. Porphins have hydrogens at all 8 substituent positions. In short, the molecule can be represented as:

HEMOGLOBIN 103

PORPHYRINS Substituted porphins are called porphyrins. Porphyrins are of two types, i.e. type I and type III. A porphyrin with completely symmetrical arrangements of substituents is called type I porphyrins whereas if the arrangement of substituents is not symmetric then it is called type III porphyrins. In nature both type I and type IlI porphyrins are found but type III porphyrins are more abundant. Porphyrins are colored compounds and show characteristic absorption spectra in both UV and visible regions. Some of the important porphyrins are: Porphyrins

1. 2. 3. 4.

Nature of the substituents at the following positions

Mesoporphyrin Uroporphyrin Coproporphyrin Protoporphyrin

where

M E A P V

= = = = =

1,2 — ME AP MP MV

3,4 — ME AP MP MV

5,6 — MP AP MP MP

7,8 — PM PA PM PM

Methyl group (—CH3) Ethyl group (—C2H5) Acetate group (—CH2COOH) Propionate group (—CH2CH2COOH) Vinyl group (—CH = CH2)

Porphyrins can form complexes with metal ions. This property is very important in their functioning in biological system. Examples: Heme is iron porphyrin, chlorophyll is a magnesium porphyrin, Vitamin B12 is a cobalt porphyrin. HEMOGLOBIN The red coloring material of blood is because of hemoglobin. It is present in RBC. Hb is globular in shape. Hemoglobin belongs to class of conjugated proteins whereas heme is the prosthetic group and globin, the protein part: Hemoglobin = Heme + Globin

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Normal adult blood contains 97% HbA1, 2% HbA2 and 1% HbF. Both alpha and beta chains have 75 percent alpha helical structure. The α-chains has 7 and β-chains has 8 helical structure. Functions of Hemoglobin 1. In the transport of oxygen from lungs to the tissues and the transport of carbon dioxide from tissues to the lungs. Hemoglobin forms a dissociable hemoglobin-oxygen complex. Hb + O2 ↔ Oxyhemoglobin 2. As buffers. The buffering action of hemoglobin is due to the amino acid histidine present in the globin part of hemoglobin. Histidine comprises 8 percent of the total amino acid make up of the globin. 3. Hemoglobin is required for both carbon dioxide and oxygen transport because these gases are only sparingly soluble in water. The presence of hemoglobin increases the oxygen transporting capacity of a liter of blood from 5 to 250 ml of oxygen. Hemoglobin plays a vital role in the transport of carbon dioxide and hydrogen ion. Myoglobin which is located in muscles, serves as a reserve supply of oxygen and also facilitates the movement of oxygen within muscle.

Significance of 2,3-Diphosphoglycerate (2,3-DPG) The stability of deoxy conformation is inceased by 2,3diphosphoglycerate in mammals. It binds electrostatically to 143rd histidine and 82th lysine in β-chains of deoxy-Hb and stabilizes T-conformation. During oxygenation 2,3-diphospho glycerate is released and T form reverts to R-conformation. Mountain sickness: When an unclemetized subject goes to higher attitudes (Hill areas/mountains) than the level of 2,3-DPG increases in the blood. This reduces the affinity of oxygen to hemoglobin liberating more and more of oxygen to peripheral tissues.

HEMOGLOBIN 105

Carbon Monoxide Poisoning Carbon monoxide has the tendency to form coordination compounds with metals, in particular with hemoglobin iron. It combines with hemoglobin to form carboxyhemoglobin (Hb CO). Hemoglobin in this form does not carry oxygen efficiently since by competiting specifically and effectively with oxygen for ferrous site0s of hemoglobin, CO can displace oxygen from hemoglobin in arterial blood. The affinity of hemoglobin for CO is approximately 210 times greater than for O2. In the lungs, hemoglobin combines with O2 to form oxyhemoglobin (HbO2) which is carried in this blood stream. O2 is released at the tissue capillary level. Since there are four heme groups in hemoglobin which can combine reversibly with 4CO or 4 O2 molecule in any combination. At the physiological pH and temperature, the combination of CO with human hemoglobin is about 10 times slower than O2. However, once formed the dissociation of carboxyhemoglobin is 210 times slower than oxyhemoglobin, which explains why the affinity of CO for hemoglobin is 210 times more than that of O2.

In lead, poisoning the RBC refer to as Howell’s Jolly bodies and Cabot ring. Heme Ferrous protoporphyrin is called heme. Heme is a chelate of ferrous iron with protoporphyrin. Heme is also called protoheme.

Synthesis of Heme The starting materials of hemoglobin synthesis are glycine and succinyl CoA.

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Structure of Hemoglobin In hemoglobin, iron is in ferrous form. When hemoglobin is converted to oxyhemoglobin, one of the linkage of iron with imidazole group of histidine in globin is replaced by oxygen. In oxyhemoglobin, iron remains in the ferrous form.

HEMOGLOBIN 107

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About 85 percent of the heme thus formed is used for hemoglobin synthesis. About 10% is used for myoglobin synthesis and the remaining 5% for cytochromes and other heme proteins. There are about 250,000 hemoglobin molecules in a single RBC. Hemoglobin molecule contains 4 heme groups combined with a globin molecule, i.e. 2 α-chains and 2 β-chains, i.e. globin part and four heme groups as prosthetic groups (one with each chain). The total molecular weight is 64, 540. globin (ferroheme)4 + 4O2 = globin (ferroheme-O2)4 Deoxyhemoglobin Oxyhemoglobin Each chain has one-heme group. One hemoglobin molecule contains four heme groups as subunits.

HEMOGLOBIN 109

Globin Globin contains 4 polypeptide chains. Two are α-chains and other two are β-chains. These four chains are arranged in tetrahedron configuration. α-chain contains 141 amino acids, whereas β-chain contains 146 amino acids. In all there are 574 amino acids in the globin molecule.

The globin moiety is formed from amino acid pool in amount of 8 gm per day in the normal adult. Thus, about 14% of the amino acids from the average daily protein intake are used for globin formation. Each α-chain has 141 amino acids whereas β-chain (also gamma and delta chains) have 146 amino acids. There are 38 histidine molecules in hemoglobin molecule. The 58th residue in α-chain is called distal histidine because it is far away from the iron atom, whereas 87th residue in alpha chain is called proximal histidine because it lies near to iron atom. The α- and β-subunits of Hb are connected by weak noncovalent bonds like vander Walls forces and hydrogen bonds. Each of the four polypeptide chains of hemoglobin has its own heme prosthetic group and iron atom. Iron contained in the heme is coordinately linked with each chain by 2 histidine residues at two imidazole nitrogens of histidine at position 58 and 87 in α-chains and 63 and 97 in β-chain of globin. The structure of oxyhemoglobin is described as R (relaxed) form and that of deoxyhemoglobin is T (tight) form. The Tconformation of deoxy Hb is maintained by electrostatic forces between carboxyl and amine groups.

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Methemoglobin Methemoglobin is a hemoglobin derivative in which iron is in the ferric form. It is also called ferrihemoglobin. Methemoglobin is dark brown in color. Conversion of ferrous to ferric iron in hemoglobin destroys its capacity to combine with oxygen and to transport oxygen. Hence, methemoglobin is useless in the transport of oxygen. Normally the conversion of hemoglobin to methemoglobin takes place in the blood but reducing substances present in red cells tend to prevent the accumulation of any appreciable amount of methemoglobin. The amount of methemoglobin present in blood is 0.3 g per 100 ml of blood. Increased amount of methemoglobin in blood gives rise to a condition called methemoglobinemia. It is caused by the failure in the normal reconversion of methemoglobin to hemoglobin or by production of methemoglobin by certain drugs. The symptoms observed in methemoglobinemia are cyanosis (blue skin) and dyspnea (labored breathing).

Hemoglobin Cooperativity The oxygenation process of hemoglobin and myoglobin is very peculiar. This can be understood in terms of a graph of fractional saturation of hemoglobin and myoglobin molecules plotted against the partial pressure of oxygen. As shown in figure, myoglobin oxygenation curve is hyperbolic whereas for hemoglobin it is sigmoidal. For myoglobin the half saturation pressure is quite low which tells us that it is a better oxygen storage molecule then oxygen carrier. The difference in the oxygenation curves between hemoglobin and myoglobin is related to their structural difference. In hemoglobin the presence of four subunits alter the nature of the oxygenation curve. As a consequence of the interplay between four subunits the binding of oxygen is cooperative. The affinity of a given heme for oxygen increases as the other heme in the hemoglobin molecule are oxygenated. Consequently the degree of saturation at first does not respond much to the pressure, then begins to rise abruptly and finally the curve levels off at high pressure. This phenomenon is called

HEMOGLOBIN 111

cooperative or allosteric effect. There is an advantage of the sigmoidal curve. The structure of hemoglobin differs in the oxygenated and deoxygenated states. The quaternary structure of oxygenated state is called the R state (for released), and the conformation of the deoxygenated state is called the T state (for tense). The ability of hemoglobin to bind oxygen decreases with an increase in acidity protons make hemoglobin dump oxygen.

Oxygenation curves for hemoglobin and myoglobin

Hemoglobin Variants Hemoglobin A1 HbA1 contains two α-chains and two β2-chains. HbA1 constituteover 98% of the total hemoglobin of the normal adult hemoglobin and is designated as α2A β2-A, or more simply α2β2. Hemoglobin A2 HbA2 contains two α2-chains and two δ2-chains. HbA2 constitute about 2 percent of the total hemoglobin in the normal adult and is designated as α2 δ2. Hemoglobin F Human fetal hemoglobin is designated as HbF and is represented as α2 γ2.

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HbF contains two α-chains and two γ-chains. HbF is predominant form present at birth but is almost totally replaced by HbA1 within few months after birth. Hemoglobin S (Sickle Cell Hemoglobin) HbS contains two α-chains and two β-chains in which glutamic acid at 6 position from the N-terminal end of the β-chain is replaced by valine. HbS is also called sickle hemoglobin due to the fact the red cells assume the shape of sickle on deoxygenation. HbS gives rise to sickle cell anemia. Hemoglobin Gun Hill It contains only two heme groups instead of four. Five amino acids are missing from the β-chain and this leads to the interference with the heme binding. In short hemoglobin variants are represented as: Chains HbA1 HbA2 HbF HbS

2α 2α 2α 2α

2β 2δ 2γ 2β glu →val at position 6

Myoglobin Myoglobin is a single polypeptide chain. Human myoglobin contains 152 amino acids with a molecular weight of 17,500. The heme is attached to 92nd histidine residue. One molecule of myoglobin can combine with one molecule of oxygen. Myoglobin has higher affinity to oxygen than that of Hb. Myoglobin has high oxygen affinity while Bohr effect, cooperative effect and 2,3-diphosphoglycerate effect can absent. The isoelectric point of myoglobin is 6.5. Bohr Effect The increase in acidity of hemoglobin as it binds oxygen is known as Bohr effect; or Bohr effect is the increase in basicity of hemoglobin as it releases oxygen.

HEMOGLOBIN 113

The effect is expressed by the equation. HHb+ + O2↔ HbO2 + H+ The above equation indicates that increase in hydrogen ion concentration will favour the formation of free oxygen from hemoglobin and conversely that oxygenation of hemoglobin will lower the pH of the solution. This reversible uptake and release of protons is responsible for the isohydric transport of carbon dioxide. The term isohydric refers to a lack of change of pH in the process. Breakdown of Hemoglobin

Bilirubin in combination with albumin reaches the liver, where it undergoes conjugation to form bilirubin diglucuronide which passes with the bile into the intestines. In the intestines, bilirubin diglucuronide is hydrolyzed, bilirubin is converted to urobilinogen. A portion of urobilinogen is absorbed from the intestines into the blood and some of it is excreted in the urine (4 mg/day). The remainder is re-excreted in the bile. The unabsorbed urobilinogen is excreted in the stool as fecal urobilinogen which is oxidized to urobilin.

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PORPHYRIA When the blood levels of coproporphyrins and uroporphyrins are increased above normal level and excreted in urine or faeces the condition is known as porphyria. Additionally reduced catalase activity has been reported in cases or porphyria. Classification

Inherited Erythropoietic Porphyria It is rare inherited disorder and is due to autosomal recessive pattern. Preponderance of type and prophyrias, both uroporphyrin type and coproporphyrin type. This is due to increased deaminase activity with isomerase deficiency. Affected individuals exhibit abnormal sensitivity to lightphoto sensitivity and develop skin lesion. Urine is usually red colored. Explanation: As uroporphyrinogen III is less formed or absent, heme formation surffers. Relative deficiency of heme produces induction of δ. ALA synthetase leading to massive production of type I.

HEMOGLOBIN 115

Hepatic Porphyria In this, there occurs abnormal and excessive production of prophyrins (chiefly type III), their precursors δ ALA and porphobilinogen. There is three types of hepatic porphyrias. 1. Acute intermittent porphyria or paroxysmal porphyria: It is autosomal dominant partial deficiency of uroporphyrinogen and synthetase. Patients present with acute attacks of abdominal pain, nausea and vomiting, constipation, CV abnormalities and neuropsychiatric signs. This is due to increased production of porphyrinogen and d-ALA. The patients do not have photosensitivity. Freshly passed urine is often normal in color but on standing in sunlight turns to red urine color. Both colorless compounds porphobilinogen and d-ALA in sunlight. Polymerases to form two colored red compounds porphobilin and porphyrin. Note: Drugs and steroids requiring cyt P-450 can precipitate acute case. Reason is excessive utilisation of cyst P-450 for which heme is utilised. This decrease in heme is associated with depression of δ-ALA synthetase. 2. Porphyria cutanea tarda: It is autosomal dominant: This is due to partial deficiency of uroporphyrinogen decarboxylase and patients are characterized by photosensitivity. Urine contains increased quantities of uroporphyrins and coproporphyrins of both types and also elevated urinary excretion of d-ALA and PBG occurs and is associated with use in serum iron. 3. Varicyate porphyria or mixed (combined) porphyria: In this neurological as well as cutaneous symptoms are seen. This is autosomal dominant. There is deficiency of portoporphyrinogen oxidase and ferrochelatase. Clinically there is vomiting, acute attacks of abdominal pain and neuropsychiatric signs and cutaneous photosensitivity.

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Bilirubin is of two types: 1. Direct bilirubin: Direct bilirubin is bilirubin diglucuronide. It is water soluble. It is expressed as conjugated bilirubin because it can be coupled readily with Diazo Reagent (diazotized sulphanilic acid). This is the direct van den Bergh reaction. 2. Indirect bilirubin: Albumin bound bilirubin is called indirect bilirubin. It is water insoluble. It is expressed as unconjugated bilirubin as it will not react until it is released by the addition of alcohol. The reaction with Diazo reagent after the addition of alcohol is called the indirect van den Bergh reaction. Normal serum bilirubin level is 0.2-0.6 mg %. Jaundice Jaundice is due to increase in the concentration of bilirubin in the blood which imparts yellow color to the skin and conjunctive. Jaundice may be either due to over production of bilirubin than what the liver can normally excrete or a damage in liver, fails to excrete bilirubin in normal amounts. Jaundice is of three types: Hemolytic or Pre-hepatic Jaundice In hemolytic jaundice, there is an increased breakdown of hemoglobin, the liver cells are unable to conjugate all the increased bilirubin formed. Increased production of bilirubin leads to increased production of urobilinogen which appears in urine in large amounts. Bilirubin will be absent in urine. Hepatocellular or Hepatic Jaundice This type of jaundice results from liver damage which cannot conjugate bilirubin. The indirect serum bilirubin level will be high. Urine will show the presence of bilirubin and increased amount of urobilinogen. Stool is light in color.

Serum bilirubin Conjugated fraction (Direct) Unconjugated fraction (Indirect) van den Bergh Reaction Urine bilirubin Urine urobilinogen

2.

3. Fecal stercobilinogen Serum cholesterol –Free form –Esterrified form

Causes

1.

Biochemical investigation

Much more than the normal Increased Normal Normal Normal

Usually diminished Decreased Decreased More than normal

Delayed direct positive Present (but in low amount) Normal

Increased

More than direct fraction Indirect positive Usually absent

Increased Increased

Disease of parenchymal cells of liver Increased Increased

Hepatocellular (hepatic)

Due to excessiv hemolysis

Hemolytic (pre-hepatic)

Biochemical changes in jaundice

Usually diminished Increased Normal Normal

Absent

Contd.

Increased (Direct form is more) Direct positive Increased

Increased Increased

Due to obstruction of biliary tract

Obstructive (post-hepatic)

HEMOGLOBIN 117

Prothrombin time Serum alkaline Phosphatase Serum transaminases Protein floculation test (Thymol turbidity test) Color of stool

5.

6.

7.

8.

*King Armstrong units.

9.

Serum proteins A:G ratio

4.

Biochemical investigation

Contd.

Dark colored

Normal or weakly

Normal

Normal

Normal

Normal Normal

Hemolytic (prehepatic)

Pale colored

usually positive

Normal or moderately increased (value below 35 KA units) Very high in first week

Decreased Decreased (Reversal of A:G ratio) Increased

Hepatocellular (hepatic)

Negative except in severe obstruction Clay colored

Moderately raised

Normal after the parenteral. Injection of vitamin K Increased (value above 5 KA* units

Normal

Obstructive (posthepatic)

118 BIOCHEMISTRY FOR STUDENTS

HEMOGLOBIN 119

Obstructive or Post-hepatic Jaundice This type of jaundice results from the obstruction of common bile duct. As a result of obstruction, bilirubin does not pass into the intestine, so no urobilinogen is found in the urine. Direct serum bilirubin level will be high, urine will show the presence of bilirubin. Stool is clay colored. Physiological Jaundice or Neonatal Jaundice Usually mild form of jaundice appears in some newborn children on the 2nd and 3rd day of life called neonatal jaundice. Causes 1. Excessive destruction of RBCs after birth causing increased in serum bilirubin. 2. Due to hepatic immaturity During IU life, bilirubin formed is mainly eliminiated by placenta immediately after birth where has to eliminate all the bilirubin but it is unable to deal adquately during first 10 days. Note: 1. In infants, when serum bilirubin rises beyond 5% clinical jaundice appears. 2. Jaundice is more common and more severe is premature babies. Phototherapy Exposure of skin to white light converts bilirubin to a compound which has shorter life than bilirubin called lumirubin. Phototherapy is used to treat infants with hemolysis.

120 BIOCHEMISTRY FOR STUDENTS

CHAPTER

6

Enzymes

ENZYMES Enzymes are biological catalysts which bring about chemical reaction in living cells. They are produced by the living organism and are usually present in only very small amounts in various cells. They can also exhibit their activity when they have been extracted from the source. Enzymes are all organic compounds and a number of them have been obtained in crystalline form. General properties of enzymes are: 1. All enzymes are proteins with exception of ribosomes. 2. Enzymes accelerate the rate of reaction by: a. Not altering the reaction equilibrium b. Being required in a very small amount c. By being not consumed in the overall reaction. 3. They have the enormous power for catalysis. 4. Enzymes are highly specific for their substrate. 5. Enzymes possess active sites at which interaction with substrate takes place. 6. Enzymes catalysis involves the transformation of enzymesubstrate complex as an important intermediate in their action. 7. Enzymes lower the activation energy. 8. Some enzymes are regulatory in function. Some enzymes are purely protein in nature and depend for activity only on their structure while certain enzymes require for their function one or more nonprotein component. They are termed as coenzymes, cofactors or prosthetic groups. If such a compound is firmly attached to enzyme proteins then

ENZYMES 121

it is called a prosthetic group. If its attachment to protein is not very firm then it is called coenzyme. Certain coenzymes exist in free state in solution and contact enzyme protein only at the times of reaction. The term apoenzyme refers to the protein part of the enzyme. The apoenzyme in combination with its prosthetic group (or coenzyme) constitute a complete enzyme or holoenzyme system. Holoenzyme = Apoenzyme + Coenzyme = Protein part + Nonprotein part Coenzymes Many enzymes in order to perform their catalytic activity require the presence of small nonprotein molecules. Coenzymes are low molecular weight, organic compounds, nonprotein, thermostable and can be separated by dialysis.

Characteristics of Coenzymes 1. 2. 3. 4.

They are stable towards heat. Generally derived from vitamins. Function as cosubstrates. They participates in: a. Hydride (H¯) and electron transfer reactions, e.g. NAD+, NADH, FMN, FAD, etc. b. Group transfer reactions, e.g. CoA, TPP, pyridoxal phosphate, tetrahydrofolic acid, etc. Coenzymes NAD+, NADP+ FAD, FMN Thiamine pyrophosphate Pyridoxal phosphate Biotin Coenzyme A

Functions performed Hydrogen transfer Hydrogen transfer Acetyl group transfer Amino group transfer Carboxyl group transfer Acyl group transfer

Most of the coenzymes are the members of water soluble B-complex group of vitamins. Coenzymes function as the

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intermediate carrier of functional groups of specific atoms or of electrons that are transferred in the overall enzymatic reactions. Classification of Enzymes According to the International Union of Biochemist, the enzymes are classified into six major classes. 1. Oxidoreductases: They catalyze oxidation and reduction reactions. These enzymes are divided into three groups. a. Oxidases: Those which use oxygen as hydrogen acceptor, e.g. tyrosinase, uricase. b. Anaerobic dehydrogenases: Those which use some other substances as hydrogen acceptor, e.g. lactic dehydrogenase, malic dehydrogenase. c. Hydroperoxidases: Those which use hydrogen peroxide as substrate, e.g. catalase, peroxidase. 2. Transferases: They catalyze the transfer of some group from one molecule to another molecule. These enzymes are important in biological synthesis, e.g. transaminases, hexokinases, transacylase, transaldolase, ketolase, phosphomutases. 3. Hydrolases: They catalyze the hydrolysis of substrate by addition of water molecule across the bond which is split, e.g. esterases, peptidases, phosphatases, deamidases. 4. Lyases: They catalyze the addition or removal of groups from the substrate without hydrolysis, oxidation or reduction, e.g. decarboxylases, carboxylase, carbonic anhydrase, aldolase, enolase, etc. 5. Isomerases: They catalyze the conversion of a compound into an isomer, e.g. racemases, epimerases, isomerases, mutases. 6. Ligases: They catalyze the linking together of molecules coupled with the breaking of pyrophosphate bound in ATP, e.g. glutamine synthetase, succinic thiokinases. Enzyme Specificity Enzyme specificity is determined by how well the reactant fit into the enzyme surface. Some enzymes are very specific and show activity with only one substrate. However, some other enzymes are much less particular and will catalyze reaction with similar compounds.

ENZYMES 123

Generally two types of enzymatic specificities are observed in different reactions. Zymogens: Several proteins are synthesized in inactive forms. These are called zymogens eq. proteins digesting enzymes and blood clotting proteins. To activate zymogens, a small amount of protein is cleared from one end. This causes the protein to change shape and activate it. These changes are not reversible.

Stereospecificity Some enzymes show specificities only with a specific group of a substrate, e.g. Urease catalyzes the hydrolysis of urea.

Alteration in the structure of urea results in the loss of activity. For example, N-methyl urea and thiourea are not the substrate for enzyme urease.

Also some enzymes show specificity towards D- and Lform of the same substrate, e.g. D-amino acid oxidase acts only on the D-form of amino acid and not on L-form.

Substrate Specificity Some enzymes catalyzes similar type of reactions but differ in their action due to absolute substrate specificity, e.g. Pepsin hydrolyzes peptide bond involving amino group of aromatic amino acids as phenylalanine or tyrosine. Similarly trypsin hydrolyzes peptide bond involving the carboxyl group of basic amino acids such as lysine or arginine.

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FACTORS INFLUENCING THE RATE OF ENZYMATIC REACTIONS Effect of Substrate Concentration At a low substrate concentration, the initial velocity of an enzyme catalyzed reaction is proportional to the substrate concentration. However, as the substrate concentration is increased, the initial velocity increases less as it is no longer proportional to the substrate concentration. With a further increase in the substrate concentration the reaction rate becomes independent of the substrate concentration and assumes a constant rate as a result of enzyme being saturated with its substrate. It was Michaelis and Menten who suggested an explanation of these findings by postulating that at low substrate concentrations, the enzyme is not saturated with the substrate and the reaction is not proceeding at maximum velocity whereas when the enzyme is saturated with substrate, maximum velocity is observed. They further visualized the combination of enzyme with the substrate to form an enzyme-substrate complex and assumed that the rate of decomposition of the substrate being proportional to the concentration of enzymesubstrate complex. The velocity of the reaction at this high

ENZYMES 125

substrate concentration is termed as maximum velocity. The substrate concentration at which the velocity is half of the maximum velocity is called the Michaelis constant and is termed as Km. Km indicates the affinity of the substrate towards the enzyme and is inversely proportional to the affinity. 1 Km ∝ Affinity Higher the affinity the smaller will be the Km and lower the affinity, the higher will be the Km. The Michaelis-Menten equation is given by the expression V0

Vmax [S] = K + [S] m

where

V0 = Initial velocity Vmax = Maximum velocity Km = Michaelis constant [S] = Substrate concentration The Michaelis-Menten equation relates the initial velocity, the maximum velocity and the initial substrate concentration through Michaelis-Menten constant. When the initial velocity is exactly half of the maximum velocity the Michaelis-Menten equation assumes the form V [S] 1 Vmax = max 2 K m + [S] Km + [S] = 2 [S] i.e. Km = [S]

Thus Michaelis-Menten constant is equal to the substrate concentration at which the initial velocity is half of the maximum velocity. Determination of important physical constants of an enzyme such as V and Km would be difficult from the curve that would be obtained by plotting [V] against [S]. So the MichaelisMenten equation can be transformed into the form which is useful in plotting experimental data. Taking the reciprocals of both the sides of Michaelis-Menten equation.

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K + [S] 1 = m Vmax [S] V0

1 Km [S] = + Vmax [S] Vmax [S] V0

or

1 Km 1 = + Vmax [S] Vmax V0 This equation is called Line-weaver Burk equation and is the equation for a straight line y = mx + c, where m is the slope of the straight line, c is the intercept on the y-axis and x is the intercept on x-axis. When

1 1 is plotted against a straight line is obtained, [V0 ] [S]'

the slope of which is the

Km 1 and has an intercept of on Vmax Vmax

1 1 1 axis and intercept of on the axis. Km [V0 ] [S]

ENZYMES 127

Since, Line-weaver-Burk equation is in the form of a straight line, so it requires few points to define, Km. By using small concentrations of substrate it is possible by this double reciprocal plot to determine Km.

Significance of Km and Vmax Values The Michaelis constant [Km] has two meanings: One is that it is equal to that substrate concentration at which half of the active sites are filled and so once the Km is shown, the fractions of sites filled (fs) at any substrate concentration can be calculated by: fs =

[S] [S] + K m

V = Vmax

Second, Km is related to rate constant of the individual steps K + K2 Km = 1 K1 Now, when the K1 is much more than K2, the K2 becomes negligible and Km is then equal to

K −1 − K1

, which is the disso-

ciation constant of the ES complex, a reversible reaction, i.e. K1

E + S ⇔ ES K−1

when or

R1 R2 R1 K1 [E] [S]

= = = =

K1 [E][S] K–1 [ES] R2 (at equilibrium) K–1 [ES]

[E] [S] K −1 = = KSE K1 [ES] (the equilibrium constant of ES) K −1 Km = K1

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So when this condition is met, Km indicates the strength of ES complex and at such conditions a high Km indicates weak binding and a low Km indicates strong binding. But this is true only when the K2 is much less then K-1. Vmax Vmax indicates the turn over number of the enzyme if the concentration of active sites, i.e. the total enzyme (Et) is known since Vmax = K2[Et]. Here, in this relation K2 is called the turn over number of an enzyme which is defined as number of substrate molecules converted into product per unit time when the enzyme is fully saturated with the substrate and the time required for each round of catalysis is thus given by 1/K2.

Method of Determining Km Km can be determined by double reciprocal Line-weaver-Burk method. In this the velocity of reaction is noted with different 1 1 and from the graph, the value of Km is determined. [S] [V ] Another advantage of this equation is that it is used to differentiate certain type of inhibitors of enzyme system. Effect of Enzyme Concentration The rate of an enzyme catalyzed reaction is directly proportional to the concentration of the enzyme. The greater the concentration of enzyme, the faster will be reaction taking place. Effect of pH Most enzymes have a characteristic pH at which their activity is maximum. Above or below that pH, the enzyme activity decreases. If a curve is drawn between the activity of an enzyme on a given substrate with the pH of the reaction mixture, it will reveal a maximum activity at a definite pH. This value is known as optimum pH. See Diagram on Page No. 129.

ENZYMES 129

This is probably due to the changes in the net charge on enzymes, (as they are protein in nature) resulting from changes in pH. Excessive changes of pH brought on by the addition of strong acids or bases may completely denature and inactivate enzymes. Effect of Temperature The rate of an enzyme catalyzed reaction generally increases with temperature, within the temperature range in which the enzyme is stable and retains its full or maximum activity. Enzyme catalyzed reactions have an optimum temperature at which the reaction is most rapid. Above this temperature the reaction rate decreases as enzymes being protein in nature are denatured by heat and becomes inactive. The increase in rate below optimal temperature results from increased kinetic energy of the reacting molecules. ENZYME ACTIVITY Activity: Amout of substrante converted to products by the enzyme per unit time (e.g. micromoles/minutes) Specific activity: Activity per quality of protein (e.g. micromoles/ minute/mg protein) Catalytic constant: Proportionality constant between the reaction velocity and the concentration of enzyme catalyzing the reaction. Unit: Activity/mole enzyme. Turnover number: Catalytic constant/number of active sites/ mole enzyme.

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International unit (IU): Quality of enzyme needed to transform 1.0 micromole of the substrite to product per minute at 30°C of optimal pH. The activity of an enzyme is expressed in standard units U = the amount of activity of an enzyme which catalyzes the transformation of one micromole of substrate per minute. The specific activity of an enzyme is the number of units of enzyme activity per mg of protein. The reason for needing this is that often the enzyme is not pure and there is contamination protein in the sample. The catalytic constant is units of enzyme activity per mol of protein (mmol/min/mol enzyme). Katab (kat) are the conversion of 1 mol/sec (International units). Turnover Number The number of molecules of substrate converted to products per enzyme molecule per minute is called turnover number. ENZYME INHIBITIONS Since, enzymes are proteins, any agent which denatures proteins will inactivate the enzyme. Inhibitors are the substances which lower down the rate of enzyme reactions. They exert their effect by acting on the apoenzyme, coenzyme, prosthetic group or activator present in the enzyme system or by interfering with the binding of the substrate to the enzyme. Reversible inhibitors bind the enzymes through noncovalent bonds and dilution of the enzyme-inhibitor complex results in dissociation of the reversibly bound inhibitor where as irreversible inhibitors occurs when an inhibited enzyme does not regain activity on dilution of the enzyme-inhibitor complex. Substances that inhibit enzymatic reactions are classified into three groups: 1. Competitive inhibition 2. Noncompetitive inhibition 3. Uncompetitive inhibition.

ENZYMES 131

This classification depends upon the manner of combination of the inhibitor with the enzyme. Competitive Inhibition As the name implies, the competition is between normal substrate and the inhibitor molecules for binding at the active site of the enzyme to form enzyme-substrate or enzyme inhibitor complex. As a result of structural similarity between the substrate molecules and inhibitor molecules, they compete both for active sites of the enzyme molecule and tie up to the active sites. These sites are then not available to the normal

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substrate molecules. The overall rate of inhibition is governed by the affinities of inhibitor molecules and normal substrate molecules for the enzyme binding site and by the concentrations of the reactants. The presence of competitive inhibitor thus increases the apparent Km of the enzyme for the substrate, i.e. causes it to require a higher substrate concentration to achieve the maximum velocity. On the other hand, a competitive inhibitor does not affect the Vmax indicating that it does not interfere with the rate of breakdown of enzyme substrate complex. Competitive inhibitors are frequently called antagonists or antimetabolites of the substrate with which they compete. The example of competitive inhibitions are: i. The inhibition of enzyme succinate dehydrogenase by malonate for succinate.

Both have similar structural resemblance and hence, both compete for the active site of enzyme succinate dehydrogenase. ii. Sulphanilamide has structural resemblance with paraminobenzoic acid and blocks the folic acid synthesis which results in the deficiency of the vitamin to microorganisms.

ENZYMES 133

In case of competitive inhibition. Affinity — Decreases Efficiency — Remains same — Decreases as Km increases 1/Km 1/Vmax — Remains same Noncompetitive Inhibition As the name implies there is no competition between the sub strate and the inhibitor molecules. There is little or no structural resemblance between the substrate and the inhibitor molecules and hence they bind to the different sites of the enzyme. Inhibitors combine with the allosteric site of the enzyme, this combination results in the distortion of the active site. In noncompetitive inhibition, the affinity of enzyme remains same but its efficiency decreases. This inhibition is also known as allosteric inhibition. The inhibitory action cannot

be overcome by increasing the substrate concentration. The complex formation between the inhibitor and enzyme is reversible. Noncompetitive inhibitors lowers the Vmax but does not effect the Km. Examples of noncompetitive inhibitions are: There are many enzymes which require free sulphydryl group (i.e.—SH group) for activity, are noncompetitively inhibited by heavy metal ions such as Pb++, Hg++, etc. Urease is an example of an enzyme which experiences heavy metal

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inhibition. The action of nerve gas poisons on acetylcholinesterase, is an example of noncompetitive inhibition. In case of noncompetitive inhibition. Affinity — Remains same Efficiency — Decreases Remains same because the substrate 1/Km concentration has no effect on the inhibitory action. — Increases as V has decreased. 1/Vmax Uncompetitive Inhibition These inhibitors combine only with the enzyme-substrate forming an irreversible complex. The inhibition is dependent only on the concentration of the inhibitor. In case of uncompetitive inhibition Vmax is lower Slope is same Apparent Km < Km

CATALYTIC SITE OR THE ACTIVE SITES OF THE ENZYMES The portion of the enzyme protein molecule which actually takes part in catalysis is called active site or the catalytic site of the enzyme. Although the enzymes differ widely in structure specifically and catalysis, there are certain common features about the active sites.

ENZYMES 135

1. Normally the active sites makes up a small volume of the total portion of an enzyme. 2. The active site is a three dimensional activity. 3. It is made up of groups that come from the different parts of the linear amino acid chain. Indeed the residues are far a part in the linear sequence but may come together to bring about catalysis. 4. The specificity of the substrate binding depends upon the precisely defined arrangement of the atoms or groups at the active site. Emil Fisher postulated that substrate and enzyme reacted in a well defined clear cut lock-key fashion signifying the predominated structure of the active fit complementary to the substrate molecule structure with which it will bind. This model implied therefore the rigidity of the catalytic site. But this hypothesis was soon found unable to explain the possibility of such a catalytic site reacting with the product to reform substrate in a reversible manner. Then Koshland proposed a more flexible hypothesis called “induced fit model” regarding the structure of the active site. According to this hypothesis, enzymes in the inactive state in the absence of substrate and that various groups in the active site are not correctly oriented to interact with the complimentary groups on the substrate. Binding of the specific substrate however, results in a conformational change in the enzyme and thus to the active site and shifting of those groups or atoms in the site into the correct position for proper binding with the substrate and catalysis. Feedback inhibition: In many multienzyme systems, the end product of the reaction sequence may act as a specific inhibitor of an enzyme at or near the beginning of the sequence, with the result that the rate of entire sequence of reactions is determined by the steady state concentration of the end product. This type of inhibition is called feed back inhibition. For example, cholesterol synthesis is regulated, by feedback inhibition. ENZYME INDUCTION Enzymes are classified according to the condition under which they are present in a cell. They are of two types.

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a. Constitutive enzymes. b. Inducible enzymes.

Constitutive Enzymes These enzymes are formed at constant rates and in constant amounts. Their presence in a cell is not related to the presence or absence of their substrates. They are considered to be part of the permanent enzymatic make of the cell. For example, enzymes of glycolytic pathway. Inducible Enzymes Also called adaptive enzymes. They are always present in trace amounts but their concentrations vary in proportions of their substrates. Isoenzymes They are multiple forms of a given enzyme having different mobilities on electrophoresis, differently depressed by inhibitors towards different substrates. Isoenzymes catalyze the same reaction but differ in Km, Vmax or both. The relative amounts of the isoenzymes of a particular enzyme differ in different organs so that in disease, different isoenzyme patterns are found according to the organs from which they have come. These forms are the characteristics of different organs and tissues of the human body. Example 1. Lactate dehydrogenase (LDH): This enzyme catalyzes the dehydrogenation of lactate to pyruvate. This occurs in five different isoenzymes. This enzyme is a tetramer having two types of units, i.e. L and M units. Depending upon the various combination, five isoenzymes are known, i.e. thest two subunits can combine in five different ways. Test

Composition

Location

LD-1 LD-2 LD-3 LD-4 LD-5

HHHH HHHM HHMM HMMM MMMM

Heart and RBC Heart and RBC Brain and kidney Liver and skeletal muscle

ENZYMES 137

LD-1 is the predominant form in heart and LD-5 in muscles. LDH is elevated from 12 to 48 hours after initial attack. 2. Alkaline phosphatase: It occurs in two forms. 3. Isocitrate dehydrogenase: It occurs in two forms. 4. Creatine phosphokinase: It occurs in three forms. CPK is a dimer consisting of one subunit found in the brain (B) and other in muscle (M). CPK is found in three isoenzymes, as CPK1 (BB), CPK2 (MB) and CPK3 (MM). In normal serum 95% of the CPK activity is in CPK3. CPK is, found in three isoenzymes as: i. CPK (MM), largely found in skeletal muscle tissue. ii. CPK (BB), predominately found in brain tissue. iii. CPK (MB), exclusively found in heart tissue. Blood level of both CPK (total) and CPK (MB) usually markedly increases following acute myocardial infarction. Only CPK (MB) elevation is highly specific for the diagnosis of MI. CPK (MM) increases rapidly following exercise or muscle trauma. CPK (BB) is heat labile and rarely detected in serum. The activitiy of CPK2 is the cornerstone for the diagnosis of myocardial infarction because of its abundance in heart and absence from other cells. It may be elevated after 4 hours and its activity may increase from two to ten folds after 16-24 weeks. DIAGNOSTIC VALUE OF PLASMA ENZYMES A determination of enzyme levels in the serum is often helpful in pinpointing which, if any, body tissue or organ has been damaged or malfunctioning. When a tissue is injured some cells of that tissue are destroyed and their contents, enzymes included, are released into the blood stream. Therefore, if an enzyme is normally found predominately in a tissue other than blood, an increase in its level in the blood indicates that tissue has been damaged. Serum acid phosphatase is increased in Paget’s disease of bone, hyperparathyroidism, metastases of bone, Gaucher’s diseases, chronic renal failure and prostatic carcinoma.

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Serum alkaline phosphatase is increased in rickets, hyperparathyroidism, obstructive jaundice, osteomalacia. Serum glutamate oxaloacetate transaminase SGOT/AST (Aspartate transaminase) is increased in myocardial infarction and skeletal muscle dystrophies. Serum glutamate pyruvate transaminase SGPT/ALT (Alanine transaminase) is increased in viral hepatitis, toxic hepatitis and other forms of liver diseases associated with some degree of hepatic necrosis. Typical profiles of serum enzymes following a myocardial infarction. SGOT catalyzes the reversible transfer of the amino group from glutamate to oxaloacetate to form α-ketoglutarate and aspartate. GOT is released from many diseased cells into serum as SGOT. SGOT is elevated in liver disease and following a myocardial infarction. The serum level has diagnostic value. It can be moderately elevated (5-fold) in people with cirrhosis and obstructive liver disease (a stone blocking bile duct). It can become very high (25-fold) in viral hepatitis. Serum lactate dehydrogenase (LDH) is increased in myocardial infarction, acute liver disease, pernicious anemia, progressive muscular dystrophies. Serum creatine phosphokinase (CPK) is increased in muscular dystrophy, myocardial infarction. Serum amylase is increased in various forms of pancreatic disturbances (Pancreatitis). Serum isocitrate dehydrogenase is increased in liver diseases, severe pulmonary infarction. Serum lipase is increased in acute pancreatitis and carcinoma. Enzyme Creatine kinase (CK-MB) Aspartate transaminase (AST) Lactate dehydrogenase (LDH)

Evidence of rise 3-6 hr 6-8 hr 12 hr

ENZYMES 139

Typical profiles of serum enzymes following a myocardial infarction.

140 BIOCHEMISTRY FOR STUDENTS

CHAPTER

7

Biological Oxidation

BIOLOGICAL OXIDATION All body reactions require energy which is obtained from chemical reactions carried out in the living cells. The stepwise oxidation of various metabolites is the principal mechanism for the liberation of energy. The utilization of oxygen and production of carbon dioxide by the tissues in the process of cellular respiration is the final phase of biological oxidation. Transfer of electrons are involved in all oxidation-reduction reactions. Every oxidation must be accompanied by simultaneous reduction and the energy required for the removal of electrons in oxidation is supplied by the reduction. The electron transport is important for the following reasons: 1. It explains how oxygen finally enter the metabolism. 2. It provides the mechanism for the regeneration of oxidation-reduction coenzymes. 3. It provides the majority of the energy derived from metabolic processes. The energy transfers involved in the oxidation-reduction systems are measured by difference in potential of various systems. Oxygen has the highest oxidation potential of the systems in the living cells and hydrogen atom the lowest. Biological oxidation is catalyzed by enzymes which functions in combination with coenzymes or electron carriers. Oxidases These enzymes catalyze the removal of hydrogen from the substrate directly to the molecular oxygen, e.g. cytochrome a3 (cytochrome oxidase), tyrosinase, uricase, etc. 2H + ½ O2———→ H2O

BIOLOGICAL OXIDATION 141

Dehydrogenases They are further divided into: a. Aerobic dehydrogenases b. Anaerobic dehydrogenases. a. Aerobic dehydrogenases: These enzymes remove hydrogen from the substrate using either O2 or artificial substance as hydrogen acceptor. These dehydrogenases are flavoproteins, e.g. xanthine oxidase, D-amino acid oxidase, catalase, peroxidases.

b. Anaerobic dehydrogenases: These enzymes use substances other than oxygen as hydrogen acceptor. These dehydrogenases are classified as: i. Pyridine nucleotides: Under this group comes nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+). The effective part which participates in the reaction is nicotinamide.

ii. Flavonucleotides: They are flavin mono nucleotide (FMN) and flavin adenine dinucleotide (FAD). The effective part which participates in the reaction is riboflavin. FAD + 2H+ + 2 e¯ —→ FADH2+ iii. Cytochromes: The cytochromes are iron containing hemoproteins in which iron oscillates between Fe++ and Fe+++ during oxidation-reduction. Various cytochromes are cytochromes b, c1, c, a.

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Oxygenases The enzymes incorporate oxygen into the substrate molecule. They are divided into two groups: a. Dioxygenases: These enzymes catalyze the incorporation of both atoms of oxygen into the substrate. A + O2 → AO2 For example, tryptophan dioxygenase and homogentisic acid dioxygenase. b. Monoxygenases: They add only one atom of oxygen into the substrate. MIXED FUNCTION OXIDASES These oxidases cause reduction of one atom of O2 and utilization of other atom for specific oxygenation of hydroxylation of the substrate. Two catalytic functions are performed by these enzymes: i. Reduction of an atom of oxygen to O¯ ii. Transfer of oxygen to the substrate. These enzymes not only require O2 but also a source of electrons, i.e. reducing agent to reduce an atom of O2 to O¯ Mixed function oxidases are metalloproteins with prosthetic group containing Fe, Cu. Examples of mixed function oxidases are: i. Phenylalanine hydroxylase ii. p-Hydroxy phenylpyruvate oxidase iii. Imidazole acetic acid oxidase iv. Phenolase complex (this enzyme is involved in the formation of melanins from tyrosine). Hydroperoxidases or Peroxidases These peroxidases catalyze the transfer of electrons from donars (substrates) to H2O2, reducing it to water. The peroxidases are specific in requiring H2O2 as electron acceptor (oxidizing agent) but various substrates may act as substrates or electron donars.

BIOLOGICAL OXIDATION 143

Catalases Catalases are specific type of hemin-containing hydroperoxidases which have the property of rapidly catalyzing the decomposition of H2O2. 2H2O2 → 2H2O + O2 Catalase enzymes are simply a specific type of peroxidase enzymes possessing very high activity toward H2O2 as a substrate but also capable of catalysing regular peroxidate reactions. Superoxide anion O¯2 is highly reactive. It is generated by a number of biological reactions including the antioxidation of quinones, thiols and reactions catalyzed by xanthine oxidase and flavoprotein dehydrogenases. Superoxides are very toxic to the cells and consequently the enzyme superoxide dismutase, present in all the cells, is responsible for protecting the cell from the harmful effect of superoxide anion, the cell in turn is protected from H2O2 by catalase Superoxide dismutase 2H++ 2O¯2 ———————————→ H2O2 + O2 Catalase 2H2O 2 ————————————→ 2H2O + O2 HIGH ENERGY COMPOUNDS High energy compounds are: 1. Acid phosphates: They are acid anhydrides of an organic acid (RCOOH) and phosphoric acid. Their general formula is:

For example 1, 3, diphosphoglyceric acid, acetyl phosphate. 2. Enol phosphates: For example, 2-phosphoenol pyruvic acid. 3. Guanidinophosphates: Two important compounds in this are creatine phosphate in vertebrate and arginine phosphate in invertebrate.

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4. Organic pyrophosphates: These compounds are ADP and ATP, GTP, IDP, etc. 5. Coenzyme A: Derivatives of coenzyme A are high energy compounds. 6. Methionione: As S-adenosyl methionine. RESPIRATORY CHAIN Transfer of electrons from substrate to molecular oxygen through a chain of electronic carriers is called electron transport chain or respiratory chain. Mitochondria contains series of catalysts called respiratory chain which are involved in the transfer of hydrogen and electrons and their final reaction with oxygen to form water. The components of respiratory chain are arranged sequentially in the order of increasing redox potential. Electrons flow through the chain in a stepwise manner from lower redox potential to higher redox potential. The amount of energy liberated in transfering electrons from one system to another is determined by difference in redox potential of the two systems. The respiratory chain is given as:

A redox potential of 0.2 volt between the components of respiratory chain results in the formation of 1 mole of ATP. The three sites of ATP formation in the respiratory chain are: 1. Between NAD+ and flavoprotein 2. Between cytochrome b and c 3. Between cytochrome a and cytochrome a3. If the substrate enters the respiratory chain through NAD+ than the ATP yield is 3. If the substrate enters the respiratory chain through flavoproteins than the ATP yield is 2.

BIOLOGICAL OXIDATION 145

Coenzyme (Ubiquinone) It is called ubiquinone because of its ubiquitous occurrence in nature. It is a lipid soluble hydrogen (electron) carrier found in mitochondrial membranes and is a benzoquinone derivative. It contains an isoprene side chain which varies from source to source. Human coenzyme Q contains 10 isoprene units. It is lipid soluble electron carrying protein and is reversibly reduced by 2H+ from FADH2. Reduced coenzyme Q is the final stage at which oxidation reaction occurs as a process of transfer of hydrogen atoms. Thereafter it is only the electrons of the hydrogen atoms which are carried down the electron transport and the 2H+ ions liberated into the medium. Other homologous of coenzyme Q contains 6 to 10 isoprene units and have been isolated from various microorganisms, e.g. chloroplasts of green plants and mitochondria of beef and other animal tissues. Cytochromes Cytochromes are electron carrier proteins containing heme. They contain protein part to which heme is attached as prosthetic group. The cytochromes undergo oxidation and reduction as a result of oscillation of iron atom with Fe++ and Fe+++ from which donates the reduced and oxidized form respectively. The five different cytochromes that has been identified in the inner mitochondrial membrane are cytochromes a1, a3, b, c, and c1. It has been found that cytochrome a and cytochrome a3 are combined with the same protein molecule to form cytochrome aa3 complex which is also called cytochrome c oxidase or respiratory enzyme. It contains 2 atoms of copper.

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Oxidative Phosphorylation Oxidative phosphorylation means that oxidation is accompanied by phosphorylation. The energy released as a result of biological oxidation is trapped in the form of high energy phosphate bonds in ATP by phosphorylation of ADP. It is divided into two groups: 1. Substrate level phosphorylation: In the substrate level phosphorylation the formation of high energy phosphate takes place on the substrate, without undergoing into the respiratory chain. The characteristics of substrate level phosphorylation are: i. Formation of ATP does not require oxygen ii. Respiratory chain does not participate iii. It is dinitrophenol insensitive. Examples Substrate level phosphorylation is best described by two examples: NAD+ (1) D-glyceraldehyde-3-PO4 + Pi + ADP ——→ PhosphoATP glyceric acid (2) Succinyl CoA + Pi + GDP → Succinic acid + GTP + CoA GTP + ADP → GDP + ATP 2. Respiratory chain phosphorylation: In this phosphorylation the formation of high energy phosphate bonds takes place as a result of transfer of hydrogen and electrons through the respiratory chain to oxygen. The characteristics of respiratory chain or electron-oxygen transport chain are: 1. It is completely inhibited by trace amounts of dinitrophenol (DNP) or by antimycin A. 2. Oxygen uptake however, is not inhibited by DNP. When a substrate is oxidized via NAD linked dehydrogenases, 3 moles of inorganic phosphates are incorporated into 3 moles of ADP to form 3 moles of ATP per atom of oxygen consumed. Similarly when a substrate is oxidized via flavin linked dehydrogenases only 2 moles of ATP is formed.

BIOLOGICAL OXIDATION 147 Type of phosphorylation

Reactions

1. Substrate level

D-Glyceraldehyde -3-PO4 Phosphoenol Pyruvate Succinyl CoA

2. Respiratory chain Phosphorylation isocitrate (electron transport chain) α-ketoglutarate Succinate

~P trapped 3-Phospho Glyceric acid

1

Pyruvate Succinate

1 1

Oxalosuccinate 3 Succinyl CoA Fumarate

3 2

P/O Ratio P/O ratio is defined as the number of inorganic phosphate taken to phosphorylate ADP per atom of oxygen consumed. Mechanism of Oxidative Phosphorylation Three hypothesis for the mechanism of oxidative phosphorylation has been postulated to account for the transfer of energy from the oxidation reductions of reactions involving electron transport chain (respiratory chain) to the synthesis of ATP. 1. Chemical coupling hypothesis 2. Chemiosmotic hypothesis 3. Conformational coupling hypothesis.

Chemical Coupling Hypothesis This is the oldest hypothesis and it postulates that the energy yielding electron transfer process is coupled with energy requiring oxidative phosphorylation through the formation of high energy intermediate compound which is generated by the electron transport system and then subsequently utilized in the ATP formation from the ADP. In effect, it proposes the existance of specific carrier proteins called C1, C2 and C3 at each of the three ATP producing sites along with an intermediate I carried by them. At the site of release of energy sufficient to form ATP, intermediate I is combined with the carrier to form a high energy carrier and intermediate

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complex. This complex then is again linked to combine with another intermediate X to form I~X again an high energy intermediate. The I component is then finally replaced by inorganic phosphates and ADP is phosphorylated to form ATP using the energy contained in I2~X complex, which is used up. A strong objection to this effect is that no such intermediate has been found even after intensive research. This hypothesis takes the help of hypothetical carriers and hypothetical intermediates I and their effects are explained as:

Effect of inhibitors: Inhibitors arrest respiration by blocking the respiratory chain at energy site I, II and III. Inhibitors of site I = Rotenone, amobarbital, piericidin Inhibitors of site II = Antimycin, BAL Inhibitors of site III = H2S, CO, CN¯ Uncouplers: Uncouplers are substances which allow electrons to continue but prevent phosphorylation of ADP to ATP. They are dinitrophenol (DNP). Uncouplers causes the hydrolysis of one of the high energy intermediates (car ~ l) resulting in the release of carrier I and energy as heat.

Chemiosmotic Hypothesis This hypothesis (Peter Mitchell) assumes two points. i. The outer mitochondrial membrane is impermeable to hydrogen ions and hydroxide ions. ii. The process goes on within matrix.

BIOLOGICAL OXIDATION 149

During electron transport, protons are released to the outside of the mitochondria. This results in the establishment of a proton gradient across the membrane, with a high concentration of protons (H+) outside the mitochondria and low concentration of protons inside the mitochondria creating an electrochemical potential difference. This electrochemical potential difference is used to derive a vectorial membrane located ATP synthetase, or the reversal of a membrane located ATP synthetase which in the presence of inorganic phosphate and ADP forms ATP.

Conformational Coupling Hypothesis According to this hypothesis (PD Bayer) the release of energy during the electron transport induces some conformational changes in the carrier protein or the coupling factor. These changes are due to the energy dependent shift in the number of location of weak bonds such as hydrogen bonds and hydrophilic interactions which normally maintain the three dimensional conformation of the proteins. Then this energy conserved in this energised conformational state is used to derive the phosphorylation of ADP by inorganic phosphorous into ATP. Simultaneously the carrier protein or the factor returns back to the original low energy conformation. This theory gets some support from the fact that inner mitochondrial membrane undergoes very rapid physical changes as the electron pass along the respiratory chain. Also this membrane shows some ultrastructural changes that accompany the addition of ADP to the respiratory mitochondria. This theory is in a way similar to the chemical coupling theory except the fact that it postulates the non-covalent bonds as the energy intermediates rather than the postulation of true high energy intermediate in the chemical theory. Shuttle System NADH is produced in the cytosol but cannot penetrate the mitochondria, i.e. the extra mitochondria NADH cannot penetrate the mitochondrial inner membrane but electron derived from it can enter electron transport chain by an indirect route called shuttles.

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Two important shuttle systems are: 1. α-glycerophosphate shuttle 2. Malate-aspartate shuttle. α-glycerophosphate shuttle: This shuttle transfers reducing equivalents from cytosol to the mitochondrial electron transport chain by the following route.

Malate-aspartate shuttle: This shuttle transfer NADH from the cytosol to mitochondria by the following route.

METABOLISM OF CARBOHYDRATES 151

CHAPTER

8

Metabolism of Carbohydrates

The major function of carbohydrate in metabolism is as a fuel to be oxidized and provide energy for other metabolic processes. In this role, carbohydrate is utilized by cells mainly in the form of glucose. It has the advantage of being cheap, easily digested and rapidly metabolized. Carbohydrate metabolism is basically the metabolism of glucose and substance related to glucose in their metabolic processes. Glucose serves as a ready source of chemical energy for humans. The sugar of blood is glucose. The digestion of carbohydrates such as starch, sucrose and lactose produces glucose, fructose and galactose which passes into blood circulation. Conversion of fructose and galactose into glucose takes place in the liver. Carbohydrates supply more than 50 percent of the energy requirement of the body. Except for ascorbic acid (vitamin C), carbohydrates are not essential to the diet, through gluconeogenesis, the body can synthesize necessary carbohydrates from certain amino acids. GLYCOLYSIS The breakdown of glucose to pyruvic acid is called glycolysis. Under aerobic condition, pyruvic acid enters mitochondria and is completely oxidized to CO2 and H2O. Whereas, under anaerobic conditions, pyruvate is converted to lactic acid. The sequence of reactions from glucose to pyruvic acid is also called Embden-Meyerhof pathway. Glucose is converted to pyruvate in 10 steps by glycolysis. Glycolysis is an extramitochondrial pathway and is carried by a group of eleven enzymes.

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Mutases are enzymes which catalyze the transposition of functional groups.

METABOLISM OF CARBOHYDRATES 153

Glucokinase is an inducible enzyme and has high km value for glucose whereas hexokinase is a constitutive enzyme and has low km value for glucose. Pyruvic acid has both a ketone or keto group and an acid group and hence it is a keto acid.

Salient Features of Embden-Meyerhof Pathway 1. The rate limiting step in glycolysis is phosphofructokinase (PFK). PFK is stimulated by fructose-6-phosphate, AMP and ADP but is inhibited by ATP and citrate. Since one of the main object of glycolysis is to produce ATP and since the presence of excess AMP, ADP or fructose-6-phosphate means that the cell is deficient in ATP. These molecules are activator of the enzyme (PFK), stimulating it to degrade more glucose and hence more production of ATP. Consequently an excess of ATP means that the cell is catabolizing more glucose than necessary; excess ATP inhibits PFK. 2. All the reactions of glycolysis are reversible except hexokinase, phosphofructokinase and pyruvate kinase catalyzed reactions because of energy barriers. 3. Enzyme enolase is inhibited by fluoride. Since erythrocytes do not have mitochondrial enzymes to oxidize glucose aerobically, they depend on glycolysis only for their energy requirement. That is why sodium fluoride (NaF) is used in the collection of blood sugar sample because it prevents glycolysis by inhibiting the enzyme enolase. Otherwise a low result will be obtained due to glycolysis.

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4. It is the major pathway by which glucose is metabolized in erythrocytes. 5. Glycolysis gives rise to certain intermediate compounds which are important for other biochemical processes. i. Glyceraldehyde-3 PO4: For triglycerides and phospholipid biosynthesis. ii. Acetyl CoA: Fatty acid and cholesterol biosynthesis. iii. Pyruvate: Alanine biosynthesis by transamination. Glycolysis has three principal features: 1. It is the degradative pathway whereby D-glucose is oxidized to pyruvate, which is further metabolized by either of the two routes. i. When the supply of oxygen is inadequate for complete oxidation, the pyruvate is reduced to lactate. ii. When the supply of oxygen is adequate (aerobic conditions) the pyruvate is oxidatively decarboxylated to acetyl CoA, which enters the citric acid cycle, where it is oxidized to carbon dioxide and water. 2. Glycolysis gives rise to certain intermediates which are common to other pathway such as pentose phosphate pathway. These intermediate compounds also provide sources of starting materials for the biosynthesis of substances such as triglycerides from glyceraldehyde-3-phosphate, L-alanine from pyruvate and glycogen from glucose-1phosphate. 3. Glycolysis is accompanied by the formation of ATP. Pasteur Effect Pasteur effect is the inhibition of glycolysis by oxygen. The rate limiting step in glycolysis, the phosphofructokinase, is inhibited by citrate and ATP. Crabttee Effect Crabttee effect is the inhibition of cellular respiration by high concentrations of glucose. This is due to the completion of glycolytic processes for inorganic phosphate.

METABOLISM OF CARBOHYDRATES 155

CITRIC ACID CYCLE The complete oxidation of acetyl moiety is effected by means of a cyclic metabolic mechanism called citric acid, also called tricarboxylic acid (TCA) cycle and Kreb’s cycle. This cycle takes place in mitochondria. The citric acid cycle operates only under aerobic conditions because it requires a supply of NAD+ and FAD which are regenerated when NADH and FADH2 transfer their electrons to O2 through the electron transport chain. TCA cycle requires the presence of oxygen, i.e. aerobic metabolism of carbohydrates and is catabolized by enzymes found in the mitochondrial fraction of the cell. Before pyruvate gains entry into the TCA cycle, it is oxidatively decarboxylated to acetyl CoA. Conversion of Pyruvate to Acetyl CoA Pyruvate is oxidatively decarboxylated to acetyl CoA by a multienzyme complex called pyruvate dehydrogenase complex. This complex enzyme system comprises of three different enzymes: i. Pyruvate dehydrogenase (29 molecule) ii. Dihydrolipoate transacetylase (1 molecule) iii. Dihydrolipoate dehydrogenase (8 molecule). which catalyze the five step reactions involved in conversion of pyruvate to acetyl CoA. The six cofactors required are (i) Mg++ ions (ii) Thiamine pyrophosphate (TPP) (iii) Lipoic acid (iv) Coenzyme A (CoASH) (v) FAD (vi) NAD+ (see page 156 for reaction). Salient Features of Citric Acid Cycle 1. Citric acid is the common pathway for the metabolism of carbohydrates, fats, and proteins; since it provides the complete oxidation of acetyl CoA to carbon dioxide and water. 2. Citrate synthetase catalyze a direct bond between the methyl carbon of acetyl CoA and carbonyl carbon of oxaloacetate. It is an irreversible reaction.

156 BIOCHEMISTRY FOR STUDENTS

3. It defines the step by which citric acid, isocitric acid, α-ketoglutaric acid, succinic acid are synthesized and degraded. The stepwise mechanism of the reactions is explained below.

METABOLISM OF CARBOHYDRATES 157

4. Many amino acids enter the cycle at several levels either at acetyl CoA, α-ketoglutarate, oxaloacetate, succinyl CoA and fumarate. 5. The rate limiting step in the TCA cycle is the conversion of isocitrate to α-ketoglutarate. The enzyme is the citrate synthetase. The availability of acetyl CoA and oxaloacetate in plenty stimulates this enzyme while succinyl CoA by competing with acetyl CoA inhibits this enzyme.

Similarly α-ketoglutaric acid, an intermediate in citric acid cycle is oxidatively decarboxylated to succinyl CoA. The enzyme involved is α-ketoglutarate dehydrogenase complex like the pyruvate dehydrogenase complex. This is an irreversible reaction forming succinyl CoA. Aresenite inhibits this reaction causing the accumulation of the α-ketoglutaric acid. Cofactors required are the same as in the conversion of pyruvate to acetyl CoA.

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Pyruvate can be channelled into TCA cycle as acetyl CoA or as oxaloacetate. This point is a switch point which controls the main function of the cycle. If pyruvate is channelled to acetyl CoA then the cycle will generate mainly energy. If pyruvate is channelled into oxaloacetate, then its main function will be to produce carbon skeletons for amino acid or fat synthesis, i.e. high levels of acetyl CoA inhibit the activity of pyruvate dehydrogenase, decreasing further synthesis of acetye CoA and the same time enhance the activity of pyruvate carboxylase, stimulating the synthesis of oxaloacetate.

The reaction is summed as:Succinyl CoA

ENERGETICS For each molecule of glucose, 2 pyruvates are formed. These are converted to 2 acetyl CoAs, each of which is brokendown to 3 NADH, 1FADH2 and 1 GTP. Hence, for 1 glucose molecule, 6 NADH, 2 FADH2 and 2 GTP are produced in the TCA cycle. Reactions Where ATP is Consumed Glucose to glucose-6-phosphate 1 Fructose-6-phosphate to fructose-1, 6-diphosphate 1

Reactions Where ATP is Generated Glyceraldehyde-3-PO4 to 1,3 diphosphoglycerate 2 × 3 = 6 1,3 diphosphoglycerate to 3-diphosphoglycerate 2 × 1 = 2 (Substrate level phosphorylation)

METABOLISM OF CARBOHYDRATES 159

Phosphoenolpyruvate to pyruvate (Substrate level phosphorylation).

2 × 1 = 2

Under Anaerobic Condition The ATP yield is 2 (Two molecules of ATP are generated in the conversion of glucose to pyruvate because NADH obtained in the glyceraldehyde-3-phosphate dehydrogenase reaction is not oxidized in mitochondria by the respiratory chain). Under Aerobic Condition Pyruvate to acetyl CoA Isocitrate to oxalosuccinate α-ketoglutarate to succinyl CoA Succinyl CoA to succinate (The substrate level phosphorylation) Succinate to fumarate Malate to oxaloacetate

2 × 3 = 6 2 × 3 = 6 2 × 3 = 6

2 × 1 = 2 2 × 2 = 4 2 × 3 = 6 ——————— Total = 30 ——————— Total number of ATP molecules formed under aerobic conditions is 38, i.e. 30 from citric acid cycle and 8 from glycolysis. Two important features of Krebs cycle are: i. Two carbon atoms enter the cycle as acetyl CoA and two carbons leave as carbon dioxide, so no net gain of carbon atom takes place. ii. The carbon atoms that leave as CO2 are not the same ones as those taken up as acetyl CoA. The tricarboxylic acid cycle or Krebs cycle serves five major functions: 1. It produces most of the carbon dioxide made in human tissues. 2. It is the source of much of the reduced coenzymes that drive the respiratory chain to produce ATP. 3. It converts excess energy and intermediate to the synthesis of fatty acids. 4. It provides some of the precursors used in the synthesis of proteins and nucleic acid. 5. Its components control directly (product precursor) or indirectly (allosteric) other enzyme system.

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This cycle is described as biochemical traffic circle, material coming to it from carbohydrate source might leave it to form fat whereas material coming to it from amino acid might leave it to form carbohydrate. The only road closed is that leading from fat to carbohydrate. Amphibolic Role of Citric Acid or Krebs Cycle Citric acid cycle is primarily a catabolic process for the final oxidation of the carbohydrates, fats and proteins into CO2 and H2O. But this cycle at the same time takes part in the various anabolic processes such as gluconeogenesis, fatty acid synthesis and amino acid synthesis by providing substrates which are the normal intermediate products of this cycle. Thus, this cycle has the dual or amphibolic role of both catalyzing the substances for energy and also taking part in synthesis. For example, the oxaloacetate and α-ketoglutarate are utilized for amino acids. Similarly, the malate and oxaloacetate are also utilized as glucose precursor in a reaction catalyzed by malate dehydrogenase first and subsequently by phosphoenol pyruvate carboxylase. Citrate is also utilized for providing acetyl CoA for fatty acid synthesis by extra mitochondrial pathway. Further succinyl CoA is utilized in the heme synthesis. Thus, all these examples establish amphibolic role of citric cycle. Hexose Monophosphate Shunt Pathway This pathway is also known as Warburg-Dickens-Lipmann pathway, pentose phosphate pathway, phosphogluconate pathway or direct oxidative pathway or reductive pathway. Though glycolysis is the principal pathway for the conversion of glucose into pyruvate in most tissues but there exists an alternative pathway. Since glucose utilization can proceed when certain reactions in the glycolytic pathway are blocked by the addition of inhibitors. Tissues where this pathway is more prominent are liver, adipose tissue, lactating mammary gland, leukocytes, testes, and adrenal cortex, etc. The enzymes of this pathway are found in the extramitochondrial cytoplasm.

Importance of HMP Shunt Pathway 1. This pathway generates NADPH, which is required in the reductive synthesis of fatty acids, triglycerides and steroids.

METABOLISM OF CARBOHYDRATES 161

2. Pentose sugars (Ribose-5-PO4) are formed which are required in the synthesis of nucleotides and nucleic acids. 3. This pathway is important in plants which synthesize glucose from CO2 by photosynthesis.

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Energetics Energy yield of hexose monophosphate shunt pathway. Although the greatest importance of HMP shunt pathway is to provide NADPH, for each carbon of glucose oxidized

METABOLISM OF CARBOHYDRATES 163

to CO2. Two molecules of NADPH are reduced or 12 molecules of NADPH per mole of glucose oxidized is produced. 12 moles of NADPH are equivalent to 36 moles of ATP. Combined aerobic and anaerobic glycolysis of one molecule of glucose gives 38 ATP, whereas HMP shunt pathway yields 36 ATP. So these two pathways of glucose oxidation are almost equivalent in energy yield. GLYCOGENESIS The formation of glycogen from glucose is called glycogenesis. Under the combined act of glycogen synthetase and branching enzyme, glucose units are added to the non-reducing ends of the pre-existing glycogen by α-(1,4) and α-(1,6) linkages to form glycogen. Glucose is phosphorylated to glucose-6-PO4, by hexokinase reaction, which is then converted to glucose-6-PO4, a reaction catalyzed by the enzyme phosphoglucomutase. Glucose-1-PO4 reacts with uridine triphosphate (UTP) to form uridine diphos-

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phate glucose (UDPG). The reaction is catalyzed by the enzyme UDPG pyrophosphorylase. Now in the presence of the enzyme Glycogen synthetase, C-1 of glucose of UDPG forms a glycosidic linkage α-(1,4) with the C-4 of the preexisting glycogen molecule. The addition of glucose from UDPG to the existing glycogen molecule takes place from the non-reducing end of the glycogen molecule, thus, permitting the origin of new glycogen molecules. When the chain has been lengthened by 6 to 11 glucose molecules, a second enzyme called branching enzyme, transfers 6 glucose molecule in α-(1,4) linkages and attaches to the nearby chain in α-(1,6) linkages, thus creating a branched point in the molecule. Branching is important because it increases the solubility of glycogen and provides a large number of non-reducing sugar terminals which are the sites of activity for glycogen phosphatase, the enzyme that breaks glycogen. Glycogen Synthetase a. Glycogen synthetase-D. It is the inactive form of the enzyme. b. Glycogen synthetase. It is the active form of the enzyme. Glycogen synthetase-D, is the dephosphorylated form. It is glucose-6-phosphate dependent, i.e. it is stimulated by glucose-6-phosphate.

METABOLISM OF CARBOHYDRATES 165

While glycogen synthetase-I is the dephosphorylated form. It is independent of glucose-6-phosphate. Glycogen Storage Diseases These are a group of inborn error of metabolic diseases in which there is an accumulation of abnormally large amount of glycogen in the tissue due to the deficiency or absence of enzymes involved in glycogen metabolism. Various type of glycogen storage diseases are given below: The classification of these diseases are based on the name of the patient first diagnosed of that disease. Type I. II. III.

Name of disease Von Geirke’s disease Pompe’s disease Cori’s disease

IV.

Andersen’s disease

V. VI.

McArdle’s disease Her’s disease

Enzyme deficient Glucose-6-phosphatase α-(1,4) glucosidase Amylo-1, 6-glucosidase, i.e. debranching enzyme. 1,4 → 1,6 transglucosylase, i.e. branching enzyme Muscle glycogen phosphorylase Liver phosphorylase

Glycogenolysis Breakdown of glycogen to glucose is called glycogenolysis. The breakdown of glycogen takes place in liver and muscle. In liver, the end product of glycogen breakdown is glucose whereas in muscle the end product is lactic acid.

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Under the joint action of phosphorylase [breaks only α-(1,4) linkages] and debranching enzymes [breaks only α-(1,6) linkages] glycogen is broken down to glucose.

The breakdown of glycogen is initiated by the enzyme Phosphorylase, which cleaves α-(1,4) glycosidic linkages starting from non-reducing end of the glycogen molecule to give glucose 1-PO4 and this process continues until four glucose residues remain on either side of the α-(1,6) branched point. Now another enzyme Glucan transferase, transfer three glucose units from one side to another, leaving a single glucose residue at the branched point followed by debranching enzyme to break α-(1,6)-linkage. The breakdown of glycogen takes place in liver and muscle. The action of liver phosphorylase and muscle phosphorylase are explained as below. Liver Phosphorylase It exists in two forms: a. Phosphorylase: It is the active form of phosphorylase. b. Dephosphophosphorylase: It is the inactive form of phosphorylase. Activation of the inactive form involves phosphorylation of the hydroxyl group of a serine residue by a specific kinase in the presence of ATP. Inactivation of the active form is catalyzed by a specific phosphatase. The action of kinase is stimulated by c-AMP which itself is formed from ATP in the presence of adenyl cyclase. Glucagon and adrenaline stimulate glycogenolysis by increasing the activity of adenyl cyclase. Muscle Phosphorylase It exists in the following forms: a. Phosphorylase a. It is the active form of phosphorylase. It is active only in the absence of 5-AMP. It is a tetramer containing 4 molecules of pyridoxal phosphate.

METABOLISM OF CARBOHYDRATES 167

b. Phosphorylase b. It is the inactive form of phosphorylase. It is active only in the presence of 5-AMP. It is a dimer containing only 2 molecules of pyridoxal phosphate. Phosphorylase a contains four molecules of pyridoxal phosphate. Whereas phosphorylase b contains 2 molecules of pyridoxal phosphate.

Phosphorylase in muscle is activated by epinephrine, which activates adenyl cyclase to form c-AMP, which stimulate phosphorylase kinase, key enzymes of glycolysis and gluconeogenesis in liver.

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Cori Cycle The cyclic process by which lactic acid is converted to glucose in liver and eventually reappears as muscle glycogen is known as Cori cycle. The Cori cycle is the body’s way of recycling lactic acid Liver Muscle

During vigorous muscle activity, muscle glycogen is converted to lactic acid. The lactic acid diffuses from the muscle into the blood stream and transferred to the liver. In liver, lactic acid is converted to glucose by gluconeogenesis. Glucose formed in this way returns to the muscle via circulation. This cycle continues and is called Cori cycle. GLUCONEOGENESIS

Gluconeogenesis is the process by which glucose or glycogen is formed from noncarbohydrate substances. The noncarbohydrate substances include glycogenic amino acids, intermediates of TCA cycle, glycerol, pyruvate, lactate, etc. gluconeogenesis is an important source for supplying glucose to various tissues when glucose is otherwise not available. Especially during

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fasting/starvation. Continuous supply of glucose is required for the functioning of brain, RBC, etc. even when food is not taken. The conversion of amino acids, lactate and glycerol into glucose takes place mainly in liver and kidney. Thus, liver and kidney are the major site of gluconeogenesis. Glucose-6-phosphatase does not exist in brain, adipose tissues or muscle. Therefore, these tissues are not gluconeogenic. Gluconeogenesis takes place when the energy requirements of the cell are at a minimal level and an energy source ATP is available. The production of glucose from noncarbohydrate precursors occurs by following pathways.

Gluconeogenesis is regulated by four key enzymes: 1. Pyruvate carboxylase. This enzymes is stimulated by acetyl CoA and inhibited by ADP. 2. Phosphoenol pyruvate carboxy kinase. 3. Fructose-1,6-diphosphate-1 phosphatase (FDPase). This enzyme is inhibited by AMP and ADP. 4. Glucose-6-phosphatase (G6 Pase). This enzyme is stimulated by inorganic phosphate (Pi) and glucose. The conversion of pyruvate to glucose is shown on the next page 171. Insulin represses the synthesis of these four enzymes whereas glucocorticoid hormones induces their de novo synthesis. GALACTOSE METABOLISM Galactose is derived mainly from lactose of the diet. Galactose is important for the formation of glycolipids and glycoprotein and for the formation of lactose during lactation.

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Important points of galactose metabolism: 1. Conversion of galactose into glucose is the main pathway. The galactose derived from the milk sugar is readily converted into glucose in the liver. 2. Conversion of UDP-galactose into UDP-glucose is a freely reversible reaction catalyzed by UDP galactose-4-epimerase. Hence, the glucose can be readily converted into galactose in the states of galactose lack, which is the way of treating alatosemia. This does not interfere with the growth. So it is not essential in the diet. 3. Galactosemia results from the deficiency of galactoseI- phosphate-uridyl transferase deficiency than galactokinase which is normal in the RBC of galactosemic patients. 4. Galactose is needed as it is a constituent of glycolipids (cerebrosides), chondromucoids and mucoprotein. 5. Galactose is also required for the lactose synthesis in the mammary gland by the enzyme lactose synthetase. Galactosemia Inability to metabolize galactose is called galactosemia. Galactosemia is an inherited disease, generally encountered in infants, characterized by inability to metabolize galactose or lactose. This results in the accumulation of galactose in the blood and ultimately excreted in the urine. Galactosemia is due to the deficiency of the enzyme galactose-1-phosphate uridyl transferase. Galactosemia gives rise to loss in weight, mental retardation and development of cataract due to the deposition of galactitol, a reduced product of galactose, in the lens. A galactose free diet avoids these difficulties and galactose that is necessary for the synthesis of cell membranes, cerebrosides, glycolipids

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and mucoproteins can be formed from glucose-1 phosphate. FRUCTOSE METABOLISM Fructose is an important source of dietary carbohydrate, accounting for approximately 20% of the total carbo-hydrate intake. Fructose is present in significant amounts in seminal fluid. It is synthesized in the prostate gland by the following

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reaction. In fructosuria, fructose is found in the urine, due to lack of enzyme fructokinase. LACTOSE SYNTHESIS Blood galactose is readily converted into glucose in liver. Here glucose is first converted into galactose by the pathway as above and then glucose and galactose combine to form lactose by the enzyme lactose synthetase.

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Failure to absorb dietary lactose is common in adults and is due to lactose deficiency and the irriability to hydrolyze lactose. Individuals with lactose deficiency can generally tolerate yoghurt (curd), a milk product. Yoghurt contains lactase that catalyzes the degration of lactose to glucose and galactose. URONIC ACID PATHWAY Biosynthesis of D-glucuronic acid takes place from glucose1-phosphate. UDP-glucuronic acid is required in detoxification reactions forming glucuronides (e.g. bilirubin diglucuronide) and in the

synthesis of proteoglycans. Also this pathway through L-uronic acid gives rise to the synthesis of L-Ascorbic acid (Vitamin C) in the animals and other plants. These reactions occur in animals and higher plants. In man, guinea pigs and other primates, the enzyme which converts L- glunolactone to 2-keto-L-gluconate is absent, thus making ascorbic acid a vitamin for them. UDP-glucuronic acid is the active glucuronic acid. It participates into the incorporation of glucuronic acid into chondroitin sulphate and other polysaccharides. Glucuronic acid conjugates with bilirubin, steroids and certain drugs for detoxification. The compound glucose 6-PO4 is at a pivotal junction to undergo various metabolic fats such as pyruvate/lactate (glycolysis), in HMP shuntpathway, in glycogen synthesis in liver and muscle, give rise to glucuronate, ascorbic acid (uronic acid pathway).

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Glucose is converted to glucose-6-PO4 by two possible enzymes depending upon the tissue. One is glucokinase (found in liver) which is highly specific for the glucose and other is hexokinase (muscle and fat cells), which catalysis the phosphorylation of most hexoses, including glucose. Pentosuria Pentosuria is characterized by the increased excretion of one or more pentoses. The pentoses normally present in urine are L-xylulose, D-ribose and D-ribulose. Essential pentosuria: It is characterized by increased excretion of L-xylulose. This is due to the deficiency of enzyme L-xylulose dehydrogenase. Other types of pentosuria include alimentary pentosuria resulting in excretion of L-arabinose and xylose due to intake of large quantities of fruits and ribosuria (due to increase in excretion of D-ribose). The patients with deficiency of glucose-6-phosphate dehydrogenase when given antimalarials like primaquine that precipitates hemolysis because G-6-PD is responsbile for the maintenance of reduced glutathione level and antimalarials produces excess of free radicals as free radicals damages the RBC’s cell membrane by oxidative stress mechanism. REGULATION OF BLOOD GLUCOSE The concentration of glucose in the blood is the net resultant of two processes. 1. Rate of glucose entrance into the bloodstream 2. Rate of glucose removal from the bloodstream.

Ways by which sugar is added to the blood 1. By absorption from the intestine 2. Breakdown of liver glycogen 3. By gluconeogenesis. The sources of gluconeogenesis are: amino acids, propionate, lactate, glycerol, etc. Ways by which sugar is removed from the blood 1. Conversion to liver glycogen 2. Conversion to muscle glycogen 3. In the synthesis of fats (i.e. triglycerides)

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4. In the synthesis of glycoproteins such as nucleic acids (nucleoproteins), lactose, etc. 5. Loss in the urine. A balance of the above two processes will keep the blood sugar level within normal limits. These two processes are influenced by a number of factors under physiological conditions.

The blood glucose level is most efficiently regulated by a mechanism in which liver, extrahepatic tissues and several hor-mones play an important part. Role of Liver Liver, being the centre of all metabolic activities is mainly responsible for the regulation of blood glucose level. In liver, exists the developed mechanism for uptake of glucose from the blood, conversion of glucose to glycogen for storage (glycogenesis), release of glucose from glycogen (glycogenolysis) and de novo synthesis of glucose from non-carbohydrate precursors (gluconeogenesis). Glycogenesis in liver can occur from blood glucose or any substance capable of giving rise to pyruvate. Due to the presence of glucose-6-phosphatase, liver glycogen can contribute directly to blood sugar (gluconeogenesis).

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Role of Extra-hepatic Tissues a. Role of muscle: Muscle glycogen does not contribute directly to the blood sugar due to the absence of the enzyme, glucose-6-phosphatase. Glycogenolysis in muscle provides glucose to blood only through the formation of lactic acid which by Cori cycle is converted to glucose in the liver. b. Role of kidney: Kidney also exerts a regulatory effect by reabsorbing glucose by the reabsorptive system of the renal tubules. When the blood glucose level rises above the renal threshold, the excess glucose appears in the urine. Role of Hormones Several hormones play an important role in the homeostatic mechanism of blood glucose level. Out of these insulin is the only hypoglycemic hormone whereas others are hyperglycemic hormones. 1. Insulin: Insulin plays an important role in the regulation of blood glucose concentration. It is secreted into the blood in response to hyperglycemia. Insulin increases the transport of glucose across the cell membranes. Insulin reduces the blood sugar level by increasing the glucose utilization by glycolysis, decreases hepatic glycogenolysis and increases glycogenesis. Hormones which keep the blood sugar level high are: 1. Epinephrine 2. Glucagon 3. Glucocorticoids 4. Thyroxine 5. Growth hormones. Mechanism by which these hormones increase the blood sugar level are: 1. By increasing the absorption of glucose from the intestines 2. By decreasing the oxidation of glucose at the tissue level 3. By preventing the synthesis of glycogen 4. By stimulating glycogenolysis 5. By potentiating gluconeogenesis. Blood sugar level is kept normal by insulin, by opposing the action of these hormones.

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2. Glucagon: Glucagon is also called Hyperglycemicglycogenolytic hormone. Glucagon is secreted by the α-cells of the islets of Langerhans. Glucagon secretion is stimulated by hypoglycemia. It causes glycogenolysis by activating liver phosphorylase. Glucagon thus counter balances the action of insulin which is secreted into the blood when the blood glucose level is high. Glucagon acts primarily on liver and does not affect glycogen breakdown in muscles. Glucagon enhances gluconeogenesis from amino acids and lactate. 3. Epinephrine: Epinephrine stimulates glycogen breakdown in liver and muscle. The stimulation of glycogenolysis is due to its ability to activate phosphorylase. Epinephrine also inhibits muscle glycogen synthesis in liver and thus directs the production of increased blood glucose. 4. Adrenal cortex hormones: Adrenal cortex secretes glucocorticoids, which lead to gluconeogenesis which is the result of increased protein breakdown and stimulation of transaminase. It also inhibits glucose utilization in extra-hepatic tissues. 5. Anterior pituitary hormones: Growth hormones and ACTH elevate the blood glucose level. Growth hormones decrease glucose uptake by the tissues, whereas ACTH stimulates the secretion of hormones of the adrenal cortex. 6. Thyroid hormone: Thyroxine has a diabetogenic action. It increases blood glucose concentration by increased absorption of glucose from the intestines. Glycosuria The excretion of detectable amounts of reducing sugar in urine is called Glycosuria. If glucose is excreted, then the condition is called glucosuria. Glucose is filtered by the glomeruli but is completely reabsorbed by the renal tubules. The reabsorption is effected by phosphorylation in the tubular cells. The maximum rate of reabsorption of glucose by the tubules (TmG—Tubular maximum of glucose) is 350 mg/minute. When

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the blood levels of glucose are elevated, the filtrate contain more glucose that can be reabsorbed, the excess passes into the urine and gives rise to glucosuria. Renal Glucosuria The blood glucose level is normal, but as a defect in the reabsorption system in the tubules, kidney threshold is lowered and glucose appears in the urine. Renal glucosuria is an example of benign glucosuria. Diabetes Mellitus Diabetes mellitus is a metabolic disorder due to the deficiency of insulin, resulting in high blood glucose level and the excretion of glucose in the urine. The most important features of diabetes mellitus are: 1. The hyperglycemia and glucosuria persist during fasting. 2. Liver glycogen falls to a low level. 3. Excretion of large quantities of ketone bodies due to increased fatty acid metabolism giving rise to diabetic coma. Diabetes or diabetes mellitus is a condition where in the body does not produce enough insulin or does not properly respond to the insulin that is produced, there by keeping glucose level in the blood high. Diabetes affects nervous digestive circulatory, endocrine, urinary system but all body system are in some way affected. There is no diabetic sure but it can only be cautted. Classification 1. Type 1 diabetes: Also called childhood onset diabetes, juvenile diabetes and insulin dependent diabetes mellitus (IDDM). Type 1 diabetes is a chronic (life long) disease that occurs when the pancreas does not produce enough insulin to properly control blood glucose levels. Type 1 diabetes can occur at any age but it is most after diagnosed in children, adolescents or young adults, In this form of diabetes, the body cannot make insulin the immune system mistakenly

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attacks the cells in the pancreas that make and release insulin. As these cells die, blood glucose levels rise. People with type 1 diabetes need insulin shots. 2. Type 2 diabetes: In characterized by the inability of the body to make insulin or properly use insulin as a result, cells can not take up or utilize glucose resulting in high blood glucose level. It is a slow onset process and person having diabetes for years without knowing. Typically with type 2 diabetes, the body still make insulin, but the cells cannot use it. This is called insulin resistance. 3. Gestational diabetes: It occurs during pregnancy, labor and delivery, women who got gestational diabetes are more likely to develop type 2 diabetes. Prediabetes That condition when a person have impaired glucose tolerance where blood glucose levels are higher than normal but not high enough to be classified as diabetes. Latest autoimmune diabetes of adults (LADA) is a condition in which Type 1 diabetes develops in adults. Adults with LADA are frequently initially misdiagnosed as having type 2 diabetes based on age rather than etiology. Maturity onset diabetes of young (MODY): Condition because of defects in β cell function. According to the latest WHO guidelines two fasting glucose blood measurement about 126 mg/dl is considered diagnostic for diabetes mellitus. People with testing blood glucose level from 100-125 mg/dl is considered to have impaired fasting glucose. HbA1c given an idea about average blood glucose control over the last 120 days. Glycosylated hemoglobin (HbA1c) test is recommended for: a. checking blood glucose control in pre-diabetes. b. Monitoring blood glucose control in diabetes mellitus. The normal value of HbA1c is 4-6% correlation between HbA1c blood glucose level.

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Correlation between HbA1c blood glucose level HbA1c

Average blood glucose level over past three months

6% 7% 8% 10%

120 150 180 240

mg% mg% mg% mg%

Higher the value of HbA1c poorer the blood glucose control >6.5 = Diabetes HbA 1c