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ISOLATION AND CHARACTERIZATION OF PROTEINS (ACID HYDROLYSIS OF MYOGLOBIN)

Group # 7, 2BMT, Mary Grace Saba, *Miguel M. Sabillena, Regine San Jose, David Santos, Virli-Anne Sebastian

ABSTRACT
Myoglobin is a vital protein for oxygen transport in vertebrates. It is present in large concentration in muscle and is responsible for the red color of the organ. Myoglobin is extracted and hydrolyzed by acid and subjected to different qualitative color tests. Paper chromatography had been done to analyze the different amino acid components of the protein.

INTRODUCTION
Proteins can be considered as polymers of amino acids. Amino acids are linked by covalent peptide bond into linear chain, which is called peptide or polypeptide chain. The properties common to all amino acids are due to the relative special arrangements of the carboxyl and amino groups. The physical and chemical properties unique to each amino acid are the result of the structure and chemical properties of the R group. Amino acids have a great variety of chemical reactive groups providing a wide range of reactivity proteins. The reactions for individual side-chain radicals and -amino and -carboxyl groups are specific both for free amino acids and proteins, whereas the reaction for peptide group is characteristic of the protein and peptides. Specific reactions are used for the purpose of identifying amino acids and proteins in biological media, for qualitative and quantitative analysis. Biuret reaction which accounts for determining peptide bonds, Ninhydrin reaction is typical for -amino acids. Xanthroproteic test detect side-chains of aromatic amino acids and Millons and HopkinsCole tests are for the determination of tyrosine and tryptophan residues, respectively. Fohls test is used to know if sulfur-containing amino acids are present and sulfosalicylic acid for the imidazole ring of histidine residues. The hydrolysis of amides under acidic condition is basically the same as protein hydrolysis, because peptide bond link is the same as an amide link. If you scale this up to a polypeptide chain, each of the peptide links will be broken in exactly the same way. That means it will end up in a mixture of the amino acids that constitute the protein. Protein hydrolysates can be tested qualitatively by different reagents and as well as paper chromatography. Partition paper chromatography is widely employed for the determination and separation of amino acids. The solvent migrates along a strip of paper and carries amino acid dissolved in it.

EXPERIMENTAL Acid Hydrolysis of Intact Protein


Myoglobin extracted from ground beef was hydrolyzed by acid after it was tested with different qualitative color tests. The hydrolysis process undergone by adding 5 mL of 8 N sulfuric acid to 0.5 grams of intact protein and then autoclaved for 5 hours (15 psi). Ten mL of distilled water was added to the autoclaved sample and then transferred to a 250 mL beaker. After the sample was transferred, 5mL of 8N barium hydroxide was added and then mixed. The sample was neutralized by adding 8N barium hydroxide or 8N sulfuric acid. It was then tested with red and blue litmus paper to check if it was already neutral. The sample was filtered and the filtrate was collected for characterization test and paper chromatography. Qualitative Color Reactions The myoglobin hydrolysate was tested with different characterization reagents namely: Biuret, Ninhydrin, Xanthroproteic, Millon, Hopkins-Cole, Sakaguchi, Fohl, and Diazo tests.

Eight test tubes were prepared for each of the test reaction. Each test tube consisted of 1 mL of distilled water added to 0.5 mL of hydrolyzed samples. In Biuret test 10 drops of the Biuret reagent was added to one test tube, shaken and the observed for color changes. For Ninhydrin test, 6-10 drops of 0.1 % Ninhydrin solution was added to the dilute sample and heated in a water bath. The appearance of a blue-violet coloration was taken note of. In Xanthroproteic test, concentrated nitric acid and concentrated sodium hydroxide 10 drops each were added slowly and then mixed. Color changes after each addition were observed. Five drops of Millons reagent was added to the diluted sample with the color taken note of. For Hopkins-Cole test, 20 drops of Hopkins-Cole reagent was slowly added to the sample and mixed well. Sulfuric acid about 20 drops was slowly added along side the inclined test tube. The color of the interface was taken note of. After the addition of 10 drops of Sakaguchi reagent and mixed, it was let stand for 3 minutes and 3 drops of 2% hypobromite was added. A red color indicated the presence of arginine in the protein. The addition of 20 drops of 6M sodium hydroxide and a few crystals of lead acetate to the diluted samples provided dark brown sediment during boiling of the sample provided the positive result in Fohls reaction. Lastly, a preparation of 3-5 drops of 1% sulfosalicylic acid with 3 drops of 5% sodium nitrite was added to 5 drops of the diluted sample and 5 drops of 10% sodium carbonate solution. A red coloration was observed. Separation and Identification of Amino Acid by Paper Chromatography A 1.5 cm margin from one edge of the paper was measure and drawn lightly with a pencil on the prepared 12 x 20 cm Whatman filter paper # 1. Ten equidistant points were labeled on the line for application of the samples. The samples were air-dried between applications by a capillary tube. The paper was then rolled into a cylinder without overlapping and then stapled. It was then placed on the pre-equilibrated chamber and then covered for the solvent to ascend. When the solvent reached at least 2 cm from the other end, the paper was removed and the solvent font was immediately marked. The paper was air-dried and sprayed lightly with ninhydrin reagent. Then it was oven-dried and observed for the amino acids that appeared as blue, purple and yellow spots on the paper.

The spot was encircled and computed for the Rf values.

RESULTS AND DISCUSSIONS


Qualitative Color Test for Hydrolyzed Myoglobin In Table 1 the characterization of the hydrolyzed protein, data results of intact protein of the same tests were compared with the former to show the difference of the reactions in characterization. Table 1. Results of Qualitative Color Reaction of Intact and Hydrolyzed Proteins Color Reaction Intact Hydrolyzed Protein Protein Biuret Blue Colorless Ninhydrin Light brown *Colorless Xanthroproteic Blue-green *Colorless Millons Flesh *Colorless Hopkins-Cole Light brown *Colorless Sakaguchi Red Flesh Fohls Black Brown Diazo reaction Red *Colorless *False results due to some technical errors in the experiment. Only the Fohls and Sakaguchi test provided a positive result for the color characterization of the hydrolyzed myoglobin indicating the presence of arginine and sulfur containing amino acid (cysteine and methionine). Compared with results of the characterization test of the intact myoglobin, only the Ninhydrin test provided a negative result indicating the absence of -amino acid in the intact protein. Paper Chromatography Analysis of Myoglobin Characterization of myoglobin using paper chromatography yielded different and incoherent results compared with the colored characterization of myoglobin hydrolysate. The Rf values of standards and the hydrolysate listed in Table 2 provided the additional protein result between the two qualitative tests.

Table 2. Rf values of the amino acid standards and hydrolyzed myoglobin. Rf values of the Spots Amino Acid Acid Standards Standards Hydrolysate Tryptophan 0.63 0.63 Arginine 0.51 0.48 Proline 0.73 Cysteine 0.36 Serine 0.63 0.63 Aspartic Acid 0.34 Tyrosine 0.72 Histidine 0.60 0.63 Glycine 0.59 0.63 Alanine 0.67 Tryptophan, arginine, serine, histidine, and glycine are separated and identified components of myoglobin. The presence of arginine in paper chromatography supported the results of the color characterization tests of the hydrolyzed protein that is characterized by the Sakaguchi test. The presence of tryptophan, serine, histidine and glycine were strengthened by the positive results of the intact myoglobin with the different qualitative colored test. Test discussion and comparison The subjection of myoglobin in acid hydrolysis which is composed of extreme pH and temperature which are common denaturing agent of protein destroyed the peptide bonds of the protein leaving the -amino acids susceptible and free for reactions. It is the primary reason why the Biuret reaction was negative for the hydrolyzed protein because Biuret reagent determines the peptide bond and quantity of a protein. Ninhydrin reaction is negative for the intact protein while positive for the hydrolyzed myoglobin due to the presence of the -amino acid in the latter. Based from resources, all of the 20 essential amino acids are components of myoglobin except for glutamine and asparagine which are not standards of the paper chromatography test. The errors of the experiment could be the cause of technical and personal errors of the experimenter. Xanthroproteic reaction is specific for cyclic amino acids such as phenylalanine, tyrosine, tryptophan and histidine. Aromatic amino acids react with nitric acid, yielding a yellow nitrocompound, which changes color to orange in alkaline medium owing to the formation of salt positive for the basic hydrolysis.

Since histidine and tryptophan are aromatic amino acids, Xanthroproteic reaction yielded a positive result for the intact protein but not for the hydrolysate caused probably by the errors or loss of protein during hydrolyzation process. Millons reagent is used for the determination of tyrosine content in proteins. It is composed of salts of mercury dissolved in nitric acid. Tyrosine (both free and constitutive of proteins) reacts with reagent and produces a purple-red salt of mercury.

Millons test is positive for the intact protein but not in the hydrolyzed myoglobin (also negative result in paper chromatography) caused probably by the same reason as in Xanthroproteic reaction. Diazo reaction is typical for proteins containing cyclic amino acids such as tyrosine and histidine. The amino acids produce a red colored diazo dye in reaction with the diazo reagent and the intensity of the color depends on the amount of cyclic amino acid present.

In the intact myoglobin, the diazo reaction provided a positive result while negative for the hydrolyzed myoglobin caused probable by the same errors and reason of the millons and xanthropeoteic reaction. Sakaguchi reaction is typical for arginine only. Arginine reacts with -naphtol and produces a red colored derivative. The presence of arginine is positive for both the intact and hydrolyzed myoglobin and supported by the presence of arginine in the paper chromatography for the hydrolyzed protein. Fohls reaction is used for determination of Scontaining amino acids. Heating of myoglobin solution in an alkaline medium leads to the formation of sodium sulfide if the protein contained sulfur amino acids such as cysteine, cystine, or methionine. In further reaction of sodium sulfide with lead acetate a dark brown colored precipitate is formed.

Rf = (distance traveled by a substance) / (distance traveled by the solvent)


Basically, components in the paper that have the same Rf values are the same amino acids. Tryptophan, arginine, serine, histidine, and glycine standards had the same Rf value with the separated acid hydrolysate of myoglobin indicating their presence in myoglobin. As compared with what the actual components of myoglobin, all of the standards are present in the protein and the negative results of the other amino acid components could be the cause of the technical aspects as with the experimenter errors.

REFERENCES
Campbell, Mary; Farell Shawn. (2008). Biochemistry (6th ed.). Canada: Brooks/Crole. The Biochemistry Department (2008). Laboratory Manual in General Biochemistry. Manila: University of Santo Tomas.

Fohls test is both positive for intact and hydrolyzed myoglobin indicating the presence of cysteine in the protein although the result of paper chromatography yielded a negative result for the hydrolyzed myoglobin. Chromatography is a technique that separates mixtures into their individual components for example: if we put black washable ink onto a tissue, the ink will spread outwards from the place where we blotted it however, the various components of the ink can't all move at the same speed as it spreads out - so the components will visibly separate. The principle involved in the separation of the amino acid components of the paper is polarity. The solvent system used (mobile phase) ethanol, water, and ammonia (80:10:10) is non-polar by nature and the more polar the side group, the farther it travelled as with proline, tryptophan, and tyrosine. The more polar amino acids aspartic acid and cysteine have the least distance travelled seen in the Rf values of the amino acid standard. The thing that is measured in chromatography is the difference between how far a substance (from the mixture) travels compared to how far the solvent travels.

Retrieved from the World Wide Web: Clark, J. (2004). The hydrolysis of protein. Retrieved January 10, 2009 from http://www.chemguide.co.uk/organicpro ps/aminoacids/proteinhydrolysis.html.

Ivanovien, L., Morkniene, R., Banien, R., Ivanovas, L. & Borutait, V. Retrieved January 10, 2009 from http://www.kmu.lt/nsc/biochemija/Labor atory_manual_PART%20I.pdf