Amino acid and Protein

COMPLETE REPORT ORGANIC CHEMISTRY 2

HERMIN HARDYANTI UTAMI (111 304 0202)

CHEMISTRY DEPARTMENT MATHEMATIC AND SCIENCE FACULTY STATE UNIVERSITY OF MAKASSAR 2012

RATIFICATION PAGE The complete report of Organic Chemistry II with the title of “Caffeine Extraction” which made by : Name

: Hermin Hardyanti Utami

ID

: 111 304 0202

Group

: VII

Class

: ICP B

Have been checked by assistant and assistant coordinator. So, this report is accepted.

Makkassar, December Assistant Coordinator

Assistant

Fandi Ahmad, S.Pd.

Muh. Jufri

Known By, Responsibility Lecturer

Dra. Hj. Ramdani, M.Si.

2012

A. Title of experiment Amino acid and Protein B. Purpose of experiment 1. Can prove the existence of a peptide bond 2. Can understand xanthoproteat reaction and biuret test against a wide - range of protein content. 3. Can understand the solubility and amphoteric properties of amino acids. C. Preview of Literature  Amino acids. 2,4-Dinitropheny deriVatsves. Thereuctron betwen 1 Fluoro-2,4-

dinitrobenzene and amino acids leade to 2,4-dinitrophenyl derivatsves. These are oftenerystalline and process relatively sharp melting points. 2,4-(NOL)2 C6H3F + NH2 –CHR- CO2H ( i ) NaHCO

3

( ii ) HCl 2,4-(NO2)2. C6H3-NH-CHR-CO2H To a solution or suspension of 0,25 gr of the amino acid in 5 ml of water and 0,5 gr of sodiumhydrogen carbunale, add a solution and exrtact with ethet (2x5 ml) to remove unchanged reagents. Pour the aqueous layer into 12 ml off cold 15 percent hydrochloric acid mith virgorous a gitatron. This mixture should be distinctly acid to congo red indicator paper. If the product separates as an oil pour try to incude crystallisation by scratching or strring. Collect the derivative bysuction

filtration

and

recrystallise

it

from

so

percent

ethanol.

(Vogel.1989:1279). You are certain to be familiar with there common, pure proteins: albumin in eggs, casenin milk and gelatin. Albumin egg while contains around 13% albumin, from latin albus, white when you shake up egg white with water, you get what looks like on almost cler solution. But this not true solution such as you get when dissolve sall or sugar. It is another kind of “solution” called a dispersion”. For more about collloidal dispersions. (Bren, Robert. 1960:99). Mechanisme for reaction amino acid ninhydrinto from folored:

“colloidal

Paper chormatography onceplayed an imprortantrole

in biochemical

analysis because it privided a method for separating, amino acids using very simple equipment. Although more modern techniques are now more commonly used, we’ll describe the principles are employed in modern separation techiques. ( Bruice.2003:969). The technique of paper chromatography separales amino acid on the basic of polarity.A few drops of a solution of a solution of an amino acid micture are applied to the bottom of strip of filter paper the edge of the paper is then placed in a solvent (typically a mixture of water acetic acid and butanol). The solvent moves up the paper by capsllaryaction, carrying the amino acids wiht it. Depending on their polaritres, the amino acids have different of finitres for mobile (solvent) and stationary( paper) phases and there fore travel up the paper at different rates. Fedding prefermences of nectarivorous ants for sugarsand amino acids were studied in an australian tropical rainforest usning artifical nectar solution .fipty

one ant species were recorded feeding on the solution. Preferences among carbuhydrates were principally concordant betwen ant species in parred tests sucrose was often preferred over fuctose, glucosa, maltose, melezitose, raffinose and xylose respectively. Attractiveness of sucrose baits in creased with concentation. (Blushgen, N.2004:155) The main determinansof anathelete’s protein needs are their training regime and habitual nutrisent intake. Most athletes ingest sufficient protein in their habitual diet. Additional proteins will confer only a minimal, albert arguably important, additional advantage. Given sufficient energy intake, lean body mass can be maintained when a wide range of range of protein intake (Tipton, K.O.2004:65). Amino acid contain two funotional group are amine group (-NH2) and carboxil group (-COOH). Amino acids nature contain amine group that bond in carbum atom -  to carboxil. 

R-CH-COOH NH3 Amino acid (Tim Dosen.2012:17) D. Apparatus and Chemical 1. Appararus

a. Test tube and rack 6 pieces/1 piece b. Measuring cup 10 ml 1 piece c. Drop pipette d. 500 ml beaker 1 piece e. wooden clamps f. stir

R-CH-COO+

NH4

amino acid in dipolar

g. spirit lamp h. Kasa asbestos and leg three i. Wash bottle j. Funnel k. Reflux l. Condensor m.rough and smooth cloth 2. Chemicals a. Glisin b. L- tirosin c. L- aspartat d. Aquadest e. Sodium hydroxide (NaOH) 10 % f. Litmus papers g. Indicator universal h. Chloride acid (HCl) 10 % dan 20 % i. Kasein j. Ice cubes k. Sodium nitric (NaNO2) 5 % l. Urea m. Matches n. Filtering Paper o. Cupper(II)Sulphate CuSO4 2 % p. Nitric acid concentrated (HNO3) E. Work Procedure 1. Solubility and amphoteric properties a. 1) Insert the test tube into the glycine 2) Add 2 ml of distilled water / aqudest into a test tube 3) Test the acidity of the resulting solution with litmus paper 4) Repeat the experiment with L-aspartic acid and L-tyrosine

b. a) Add 1 mL of 10% NaOH solution to the suspension of 0.1 grams of L tyrosine in 2 mL of water, and record the results. b) Insert a piece of litmus paper into the solution and menambahkansetetes by drop until the solution is acidic started. c) Stir for 1 minute and then observed and recorded the results. d) Add 10 drops of the acid solution again, then observed. c. a) Enter 0.1 grams of casein (milk protein) into a test tube. b) Add 5 ml of distilled water and 2 mL of 10% NaOH laruta. c) Close the test tube and shake until a colloidal solution. 2. Reaction with nitrous acid 1. a) Enter 0.1 grams of glycine into a test tube b) Add 5 ml of 10% HCl solution c) In another test tube, add 5 ml of 10% HCl solution for comparison. d) Cooling the second test tube to ice water. e) Adding carefully 1 ml of 5% NaNO2 solution. Into each test tube and recorded the results. 2. Cooling the casein solution was prepared in ice water, then add 1 ml solution of NaNO2. 3. Biuret test 1.

a) Include 0.5 g urea into a dry test tube b) Heat slowly until the urea melt and gas is formed. c) Mecatat gas odor is tested with litmus paper wet mouth tube. d) Continue heating until the formation of gas stops and the rest from solid. e) Cool the solution, and then dissolving the solid in hot distilled water. f) Filter the solution and add to the filtrate 2 ml of 10% NaOH solution, then 2 -3 drops of 2% CuSO4 solution. g) Stir the solution and observe the color. h) For comparison, dissolve 0.5 grams of urea in 3 ml of water. i) Add 2 ml of 10% NaOH solution j) Then add 2 -3 drops of 2% CuSO4 solution, and then compare the results with the above observations.

2. a) Include 2 ml of distilled water in a 2 ml casein solution provided (1.c) b) Then add 2 drops of 2% CuSO4 solution Stirring and observe. 4. Xantoproteat test a. Placing 0.1 grams of casein into a test tube. b. Add 2 ml of concentrated Nitric Acid c. Heats slowly d. Observing the color that occurs e. Cooling the reaction mixture f. Neutralize carefully with 10% NaOH solution, then add a bit of excess base. g. Take note if the color of the solution changes. 5. Hydrolisis Protein a. Entering 0.5 grams of casein into a test tube. b. Adding 2 ml of 20% HCl solution, then heated above the spirit lamp. c. Cooling the reaction mixture to room temperature. d. Cooling the partial hydrolysis results in ice water. e. Comparing the results with the experiment above f. on the other, neutralizing with 10% NaOH. g. Add 3 ml of 10% NaOH solution and 2 drops of 2% CuSO4 solution. h. Preheat over spirit lamp, and then compare the results with the experiment above. F . Observation result 1. Solubility and amphoteric properties a. Solubility test 1) 2 ml water (transparant) + glisin (white)→netral and soluble 2) 2 ml water (transparant) + tirosin (white)→acid pH=5 and not soluble 3) 2 ml water (transparant) + aspartic acid (white)→acid pH=3 and less soluble b. Amphoteric test 1. 2mL aquades + 0,1 g L-Tirosin (white)+ 1ml NaOH 10%→yellow transparant→base→HCl 10% 50 drops→yellow→stir→10 drops HCl 10%→acid solution pH=2.

2. 0,1 g kasein + 5mL aquades→transparant solution + 2mL NaOH 10%→transparant solution 2. Test with nitrous acid a.1) 0,1 g glisin + 5mL HCl 10% solution+ 1mL NaNO2 5% 2) 5mL HCl 10% -

transparent solution

cooling 0ºC

transparent

transparent solution and bubbles

cooling 0ºC

transparent solution + 1mL NaNO2 5%

transparent solution and not bubbles

b. 0,1 g kasein + 5mL aquades

transparent solution and nit bubbles gas.

3. Biuret test a. 0,5 g urea

heat

transparent solution→litmus(base)→transparant solution+hot

water→ white precipitate → filter → filtrate + NaOH 10%→transparant solution+CuSO4 2% 2 drops (blue) → purple solution Compare : 0,5 g urea + 3mL aquades → transparent + NaOH 10% 2ml → transparent + CuSO4 2% 2 drops (blue)

blue solution

b. 0,1 g kasein + 2mL aquades

transparent solution+ 2 drops CuSO4 2%

→blue solution. 4. Xantoproteat test 0,1 g kasein + 2mL HNO3 concentrated (transparent) → heated → transparent + NaOH 10% until netral + indicator universal → netral → base solution (hot) 5.Hydrolysis protein 0,5 g kasein + 20 mL HCl 20% concentrated → transparant → reflux 40 minutes→18 ml (turbid)→divide 2 part 1st part → 9 ml +NaOH 3ml → yellow + 2 drops CuSO4 2% → purple solution. 2nd part → cool down + NaOH 20 % → yellow solution + CuSO4 2% 2 drops →purple solution G. Discussion 1. Solubility and amphoteric properties Amino acid that used are glysin, L-aspartic acid and L-tyrosine in water solubility. Water easy solute in water because not have alkil group or aril group only hydrogen because free group amin bigger than carboxyl then both of amin

group and carboxyl in amino acid will be act each other result zwitteren ion. Because of that, dipolar structure then amino acid soluble in water. H CH COOH + H2O

COO-

H CH NH3+

NH2 glysin

Aspartic acid get the pH is 3 and difficult soluble in water. L-aspartic acid is acid because contain two carboxyl group. Reaction: HOOC -

OOC

CH2 CH2

CH2

COOH + H2O

CH COO+

NH3 L tyrosine is acid with pH is 5 and not soluble in water because R in amino acid is amphoteric. Amino acid is non esensial amino acid that have benzene ring(aromatic). L-tyrosine is acid because ion H+ that disosiation in solution from carboxyl group. Reaction : COO-

COOH H2N

C

H

+ H2O

H3N+

C

CH2

CH2

OH

OH

H + H+

Amphoteric test used L tyrosin and casein as sample. L-tyrosine added with water and NaOH 10% produce yellow solution. The function of NaOH is to base the solution, then add HCl to acid the solution. It is same with theory that amino acid react with acid and base condition (amphoteric properties).

Reaction: COOH H2N

H2N

C

H

COONa + NaOH

H2N

C

H + H2O

CH2

CH2

OH

OH

COONa

COOH

C

H

+ HCl

H2N

C

H + NaCl

CH2

CH2

OH

OH

Cassein soluble in water get transparent solution then added NaOH 10% to soluble casein and give acid condition, it is showed casein easy soluble in water. Reaction : H2N

CH C NH CH C NH CH C OH + H2O R

H2N

O

R

O

R

O

CH C NH

CH C NH CH

C O- + H+

R

R

O

O

O

R

2.Reaction with nitrous acid Glysin crystal reacted with HCl and NaNO2 produced transparent solution and bubbles, it is showed that amino acid can reacted with NaNO2 produced N2 gases because free amino acid group. Reaction : H2N-CH2-COOH+NaNO2→OH-CH2-COOH+H2O+N2↑ Compare solution without glysin crystal get transparent solution and not bubble. It is because reaction HCl and NaNO2 not formed N2 gas. Reaction: HCl+NaNO2→HNO2+NaCl 2HNO2→H2↑+NO2 The reaction of casein with NaNO2 solution produced clear solution and there is no gas bubbles, it is proved that casein did not react with nitrous acid as amine-free casein do not exist. O O || || -NH-CH-C-NH-CH-C-O| | CH2 CH2

OH

OH

+NaNO2 →

n

3. Uji Biuret In this experiment, urea is main sample. Urea heated until formed transparent solution and gases. Gases that produced is NH3 that stinky smell. Reaction: H2N

C O

NH2 + H2N + H2N C N2H O

Urea H2N

C NH C NH2 + NH3 + CuSO4 O

O

then added water that have done heated to soluble urea produce white precipitate then filter and we get transparent solution, then added NaOH to give base condition in solution to change the color and avoid precipitate CuSO4 and break the peptide bond. Then added with CuSO4 produce blue prusi solution that sign exsistence of peptide bond. Because of it, urea heated more than melting point then urea will be change became biuret that reacted with CuSO4 produce purple solution. Reaction: H2N-C-NH-C-NH2 + CuSO4 → || || O O Biuret H3N

NH3

C=O

C=O

NH

Cu2+

NH

C=O

C=O

NH3

NH3

complex ion purple solution

Comparing, urea solution (without heat) added water produce transparent solution then added NaOH and CuSO4 produce blue solution, it is mean that not peptide bond. It is because when adding CuSO4 not reacted with polypeptide that arrange protein form complex that purple. Casein that reacted with water and CuSO4 produce purple solution it is mean that peptide bond because casein is protein that arrange from amino acid.

OH

+ CuSO4 → CH2 HC-NH2 O=C-O- Cu- O-C=O n

4. Xantoproteat test The aim from this experiment to identify exixtence of benzene group in protein. The principle is nitration benzene nuclear by nitric acid concentration and produce orange solution. In this experiment, we get transparent solution casein, it is not same with theory because mistaken of apperantice. The function HNO3 as cause occur nitration reaction because the nuclear benzene from amino acid will reacted with HNO3 and produce yellow solution. The function of NaOH as clearly existence of benzene ring because in base condition.

Reaction: COOH H2N

C

H

COOH + 2 HNO3

CH2

H2N O2N

OH

n

C

O2N CH2

H + NaOH NO2

OH

H

+ H2O

CH2 NO2

OH

COOH H2N

C

n

COOH H2N C O2N

H

+ H2O

CH2 NO2

OH

5. Protein hydrolisis In this experiment, casein added with HCl 20% then reflux belong 40 minutes to break peptide bond of protein until decompetition become amino acid. The product of reflux is turbity solution. Residue solution divide two part, 1st part cold and added NaOH 20% produced yellow solution then added CuSO4 and produced purple solution. Others solution added with NaOH and CuSO4 and produce purple solution. It is not same with theory because mistaken apperantice when do the reflux and adding some reagent and because purple sign still contain peptide bond and casein not hydrolysis.

Reaction is :

+ H2 O

→

tyrosin casein H. Closing 1. Conclusion a. Glycine soluble in water and is acid, L-aspartic acid and is slightly soluble, Ltyrosine is insoluble in water and acidic properties of casein and insoluble in water and acidic. b. In the biuret test, peptide bond is indicated by the color change to purple. c. Xantoproteat Tests showed benzene groups on the protein and amino acids (casein and L-tyrosine) are marked with orange color solution. d. Amoni acid reaction with nitric acid to release N2 gas is characterized by the presence of gas bubbles. e. Hydrolysis of proteins can lead break peptide bonds that protein breaks down into its constituent amino acids. 2.Suggestion Practitioner should know the nature and structure of the amino acid used. It is expected that the next practitioner to be more careful and meticulous in carrying out experiments to minimize errors obtained more satisfactory results.

BIBLIOGRAPHY Bluthgen, N. 2004. Preferences of Sugars and amino acids and their conditionality in adiverse nectar-feeding antcommunity. Germany. Journal of Animal Ecology Brent, Robert. 1960. The Golden Book of Chemistry Experiments. New York. Golden Press. Bruice. 2003. Organic hemistry 4th Edition. New York. Logman. Tim Dosen Kimia Organik.2012. Penuntun Praktikum Kimia Organik II. Makassar: Jurusan kimia FMIPA UNM. Tipton, K.D. 2004. Protein and Amino acids for athletes. USA. Journal of Sports Sciences. Vogel. 1989. Practical Organic Chemstry 5th Edition. New York. Longman Group.

Answer Question 1. Give the molecular formula of glycine, aspartic acid, tyrosine, and describe the acidity, alkalinity, and neutrality of the solution in water.. H2N CH COOH HCH2

H2N CH

COOH H2N CH COOH

CH2

glysin

COOH aspartic acid tyrosin

glycine has a dipolar structure in which both amine and carboxyl groups will interact generate zwitter ion solubility in water so nearly neutral. Aspartic acid has R group consisting of carbon atoms and has many carboxyl group to another so the solution is acidic. Tyrosine is an amino acid that has a dipolar structure in which both amine and carboxyl groups interact with each other so that the zwitter ion produced nearly neutral solution in water. 2.Write the equation that can explain what happens when the solution is slowly acidified.

COONa H2N

C

H

COOH + HCl

H2N C

H + NaCl

CH2 CH2

OH

OH

3. Explain the differences in the nature of hydrolysis of casein with the results of the nitric acid and the biuret test a. The difference in the nature of casein by hydrolysis to yield nitric acid, formed here N2 gas wave that signifies the hydrolysis of peptide bonds of protein polymers.

b. The difference in the nature of the results of hydrolysis of casein with the biuret test, still in the form of casein protein as peptide bond which is marked by a purple discoloration of the solution, the color formed is caused by the formation of complexes between Cu 2 + to the N atom of the peptide molecules. 4. Suggest an explanation of "aspartic acid will move more slowly than phenylalanine", the paper chromatography experiment. On paper chromatography experiment, aspartic acid will move faster than phenylalanine as aspartic acid has a smaller molecular size compared to the size of molecules that aspartic acid phenylalanine easily absorbed by the pores of the filter paper.