ISOLATION, ENZYMATIC HYDROLYSIS AND CHARACTERIZATION OF MYOGLOBIN

EXPERIMENT NO. 2: ISOLATION, ENZYMATIC HYDROLYSIS AND CHARACTERIZATION OF MYOGLOBIN Jill Cristine G. Genuino, Herald Jervy D. Go, Jianna Nadine B. Gonzalez, Gonzalez , Luke Gian C. Guerrero, Paolo F. Gutierrez, and Bienn Paulo Laforteza Group 4 2G Medical Technology Biochemistry Laboratory

ABSTRACT Proteins are large molecules consisting amino acid chains which our bodies and the cells in our bodies need to be able to function properly. Our body structures, functions, the regulation of the body's cells, t issues and organs cannot exist without proteins. Our muscles, skin, bones and many other parts of the body contain significant amounts of protein. Protein accounts for about twenty percent percent of our total body weight. This part of the experiment was was performed following the proper isolation of the protein myoglobin myoglobin from the beef muscle by process of grinding. Myoglobin is important to oxygen oxygen storage storage in living organisms. organisms. It is especially especially found in muscles and it gives it it its red colour. colour. The mechanism used in the enzymatic hydrolysis of myoglobin is a naturally occurring enzyme which is usually used by the body to digest proteins proteins in food. These enzymes enzymes are called proteases, proteases, peptidases, peptidases, or proteolytic enzymes. enzymes. They cleave the peptide bonds of proteins at specific amino acid residues, thereby separating and dividing the protein into smaller amino acid chains and some free amino acids. This experiment was was performed with with the use of a protein mixture, saturated protease solution, a phosphate buffer with pH 7.5, and incubation at 35-40˚C. The effect of the protease on the myoglobin was evident, as it turned the solution with cloudy brick-red residue into a clear solution with cloudy sediments at the bottom.

INTRODUCTION Proteins are large molecules consisting amino acid chains which our bodies and the cells in our bodies need to be able to function properly. Our body structures, functions, the regulation of the body's cells, tissues and organs cannot exist without proteins. Our muscles, skin, bones and many other parts of the body contain significant amounts of protein. Protein accounts for about twenty percent percent of our total body body weight. They are the most abundant organic molecules found in living cells. Myoglobin is a protein commonly found in muscle tissues. It is characterized characterized by its cloudy reddish coloured coloured residue and, functioning similarly to hemoglobin found in blood, it carries and stores oxygen for proper muscle function. It is the reason reason why muscle tissues appear appear to have a reddish tinge. Myoglobin is an unusual protein as it is made up almost exclusively of α-helices joined by short loops. Most proteins proteins have both α -helices and βpleated sheets.i Myoglobin can be isolated by ammonium sulfate precipitation from the buffered muscle extract.ii Meanwhile, the enzyme that was used to perform the enzymatic hydrolysis on the protein myoglobin is called protease. Proteases (also called peptidases or proteolytic enzymes) occur naturally in all organisms. Simply defined, protease catalyzes the hydrolytic breakdown of proteins. iii Protease refers to a group of  enzymes whose catalytic function is to hydrolyze (breakdown) peptide bonds of proteins. They are also called proteolytic enzymes or proteinases. Proteases differ in their ability to hydrolyze various peptide bonds. Each type of protease has a specific kind of peptide bonds it breaks. Examples of proteases include: fungal protease, pepsin, trypsin, chymotrypsin, papain, bromelain, and subtilisin.iv

Myoglobin can be isolated from the meat by ammonium sulfate precipitation from the buffered muscle extract. The experimentation of enzymatic hydrolysis and the analysis of the products are done to determine and extract more information about their amino acid compositions. The twenty amino acids are are commonly commonly found as hydrolysis products of proteins because, as mentioned earlier, enzymes catalyze the breakdown of  myoglobin. The product of this enzymatic hydrolysis of  myoglobin was stored to be used for other experimental procedures. Examples of such tests are the following: following: the tests for qualitative color reactions, separation and identification of amino acids by thin layer chromatography, and protein assay using the Bradford Method. The qualitative color reactions involve several tests such as the Biuret test, the Ninhydrin test, the Xanthoproteic test, Millon’s Test, the Hopkins-Cole test,

the Sakaguchi test, the Nitroprusside test, Fohl’s test, the test for amides, and the Pauly test. The separation and identification of amino acids by thin layer chromatography can be used for the qualitative analysis of the amino acid constituents of the acid, alkaline, and enzymatic protein hydrolysate. The separation in this type of chromatography is based on the polarity of the solutions. The Bradford Assay is commonly used to determine the total protein concentration of a sample. The method is based on the binding of Coomassie dye to proteins in acidic solution leading to an increased absorbance of the sample at 595 nm. The assay is sensitive to about 20 to 200 µm protein.

EXPERIMENTAL A. Compounds Tested (or Samples Used) For Isolation of Proteins Minced beef muscle (NH4)2SO2 70% buffer diluted (NH4)2SO2 solution, pH 7.5

For Enzymatic Hydrolysis Isolated myoglobin Saturated protease solution

For Enzymatic Hydrolysis

For Qualitative Color Reactions

1. Prepare 1g/100 protein mixture

6 M NaOH 3 M NaOH 2.5 M NaOH 20% NaOH 10% NaOH Conc. NaOH 5% NaNO2 10% Na2CO3 2% Nitroprusside solution 0.02% Naphthol solution 1% Sulfanilic acid 0.1% Ninhydrin solution Conc. HNO3 Conc. H2SO4 Hopkins-Cole reagent

distilled

water

The protein is diluted by adding one part of protein to 9 parts of distilled water, giving it a 9:1 ratio, involving taking one part of the sample and addition it to 9 parts of distilled water. In this case the dilution factor is 10. This procedure is continued until the ratio of  1:1000 is achieved. Several test tubes, a graduated cylinder, and a pipette were used by the group to perform this dilution.

2. Mix 10 mL of protein mixture and 10 mL of saturated protease solution

2% NaOBr 0.1 M CuSO4 5% (Ch3COO2)Pb

For Separation and Identification of  Amino Acids by Thin Layer Chromatography 2% w/v tryptophan 2% w/v arginine 2% w/v proline 2% w/v cysteine 2% w/v serine 2% w/v aspartic acid 2% w/v tyrosine 2% w/v histidine 2% w/v glycine 2% w/v alanine Acid, basic, and enzymatic hydrolysates 1-butanol:acetic acid:water (4:1:5) 1% Ninhydrin solution in spray bottle

Assay

mL

Before undertaking this dilution method, one must first know how to compute for dilutions.

Millon’s reagent

For Protein Method

Place 6.0 g minced meat beef heart (or steak) and 6 mL 70% (NH4)2SO4 solution in a small beaker. Gently stir the mixture for one minute to release the myoglobin. Express the dark-red extract into a new beaker using cheesecloth. Centrifuge the extract at 13,000 x g for 5 minutes. Transfer 1.5 mL of the supernatant into another empty centrifuge tube. Add 0.30-0.35 g (NH4)2SO4 crystals ground to fine powder. Mix gently until the solid dissolves. Avoid frothing. Centrifuge sample again for at least five minutes. Decant off supernatant, describe the appearance of purified myoglobin residue.

Using

Bradford

Bradford reagent Bovine serum albumin (BSA) standard (10 mg/mL) Evaporated milk sample

B. Procedure For Isolation of Proteins

An assigned group prepared the saturated protease solution. 10 mL of protein mixture was added to the 10 mL saturated protease solution inside an ignition tube.

* Alternatively, 0.050 g of protease may be added directly to 50 mL protein mixture This was not performed by the group.

3. Add 10 mL 0.1 M phosphate buffer at pH 7.5 This was added into the ignition tube as well. The function of the buffer is to maintain a regular pH within the solution. This is because the solubility of the proteins can be altered merely by changing the pH of their environment. The buffer was measured from the volumetric flask which contained it by using a 10 mL pipette. It is very important to note that when dealing with colorless liquids, the lower meniscus should be taken into account, whereas if the liquid is colored, the measurement should be based on the upper meniscus.

4. Incubate the hard glass tube in a water bath maintained at 35-40˚C for 60 minutes

In this part of the procedure, the water bath to be used depends on the source of the enzyme.

* Alternatively, the digestion of  myoglobin may be carried out overnight at room temperature 5. Allow the mixture to cool off before using it in the procedures for qualitative color reactions and for the separation and identification of amino acids by thin layer chromatography.

For Qualitative Color Reactions For each test, prepare in separate test tubes an intact protein solution (0.50 g of the myoglobin in 1.0 mL distilled water) and 0.50 mL of the hydrolysed sample. BIURET TEST Add 20 drops of 2.5 M NaOH to the samples. Mix well. Add 2-3 drops of 0.1 M CuSO4 solution. Shake the test tube and note the color of the solution. NINHYDRIN TEST Prepare 6-10 drops of 0.1% ninhydrin solution into the diluted samples. Heat the tube in a boiling water bath. Take note of the appearance of a blue-violet coloration. XANTHOPROTEIC TEST Slowly add 10 drops conc. HNO3 to the diluted samples. Mix and note the color of the solution. Slowly add 10 drops conc. NaOH. Mix and note again the color of the solution.

MILLON’S TEST Add 5 drops Millon’s reagent to the diluted samples. Note the change in color. HOPKINS-COLE TEST Slowly add 20 drops Hopkins-Cole reagent to the samples. Mix well Incline the test tube and add slowly along the side 20 drops conc. H2SO4. Do not shake the mixture. Note the color produced. NITROPRUSSIDE TEST Add 0.5 mL of 3 M NaOH to 0.5 mL of sample. Add 0.25 mL 2% nitroprusside solution. Note the formation of a red solution.

FOHL’S TEST Add 5 drops 30% NaOH and 2 drops 5% (CH3COO)2Pb to the samples. Place the tube in a boiling water bath. Note the appearance of dark (black or brown) sediment. TEST FOR AMIDES Add 1 mL of 20% NaOH to 10 drops of the sample. Place the tube in a boiling water bath. Test for the evolution of gas during heating by placing a moistened red litmus paper over the mouth of the tube. Take note of the result. PAULY TEST Prepare the diazo reagent by mixing 3-5 drops 1% sulfanilic acid with 3 drops 5% NaNO3 solution. Add 5 drops of the sample and 3-5 drops 10% Na2CO3 to the diazo reagent. Note the appearance of a red coloration.

For Separation and Amino Acids Chromatography

Identification of  by Paper

Draw the origin as pencil line across the paper with a 1.5 cm margin from the bottom of the longer edge. Mark thirteen equidistant points for spotting. Apply standards 5 times and samples 10 times. Place the paper inside a pre-equilibrated chamber. The lever of the solvent should be below the origin. Cover the chamber. It is important that the solvent be absorbed by the paper undisturbed. Air-dry chromatogram and spray lightly with 1% ninhydrin reagent. Place chromatogram inside an oven (group used hot plate istead) and amino acids will appear as blue, purple or yellow spots.

For Protein Method

Assay

Using

Bradford

Prepare a series of test t ubes as follows: Tube No. 1 2 3 4 5 6 7 8 9

mL Standard 0 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45

mL H20 1.5 1.40 1.35 1.30 1.25 1.20 1.15 1.10 1.05

Dilute the milk sample 1:500, 1:1000, and 1:2000 with distilled water. Take 1.5 mL of diluted and label them as numbers 10, 11, and 12. Add 1.5 mL Bradford reagent to each test tube. Mix well. Let it stand for 5 minutes. Read the absorbance at 595 nm within the hour. Use tube 1 as the blank.

RESULTS AND DISCUSSION Figure 1. Structure of Myoglobin

Isolation of the protein myoglobin resulted in a milky yellow curd. Enzymatic hydrolysis of the isolated protein yielded to a clear solution with sediments.

Due to the lack of time, the group was not able to finish the paper chromatography, thus the ascension of the solvent only reached half way.

Table 1. Results of Qualitative Color Reactions with Enzyme Hydrolyzed Samples

References

Test Biuret Test Ninhydrin Test Xanthoproteic Test

http://www.massey.ac.nz/~wwbioch/Myoglobin/Mb_frameset. htm ii Laboratory Manual in General Biochemistry iii http://medical-dictionary.thefreedictionary.com/protease iv http://www.enzymeessentials.com/HTML/proteases.html

Millon’s Test Hopkins-Cole Test Sakaguchi Test Nitroprusside Test

Fohl’s Test Test for Amides

Pauly Test

Reaction (+) Blue upper layer (+) Violet solution (+) Turbid solution (+) Turbid solution (+) Yellow layer (+) Colorless solution (+) Yellow solution (+) Yellow solution, brown sediments (+) Yellow layer, litmus paper is red to red (+) Pale yellow

The biuret test is used to detect the presence of  peptide bonds while the Ninhydrin test is a typical test for an α-amino acid. The Xanthoproteic test detects side

chains of aromatic amino acids while the Millon’s and Hopkins-Cole tests determine tyrosine and tryptophan residues, respectively. The Nitroprusside test is used to find out if sulfur-containing amino acids are present; test for amides it used to detect R-groups of asparagine and glutamine.

Figure 2. Paper Chromatography of  Acidic, Basic, and Enzymatic Hydrolyzed Samples and Amino Acids

Acidic

Basic

Enzymatic

Amino Acids

i