Qualitative Analysis of Myoglobin and Its Hydrolysate (1)

QUALITATIVE TESTS OF INTACT PROTEIN AND HYDROLYZED PROTEIN OF MYOGLOBIN ABSTRACT Chemical analysis is the act of decomposing a substance into its constituent elements. Chemical analysis can be either quantitative or qualitative in nature. Quantitative chemical analysis, branch of chemistry that deals with the determination of the amount or percentage of one or more constituents of a sample. On the other hand, in qualitative chemical analysis we are not concerned with the quantity of each substance, but only whether certain substances are present or absent. The objective of this experiment is to isolate myoglobin from beef heart by salt-induced precipitation and to analyze chemical groups responsible for color reactions and explain the principle involved in each test. The intact proteins and hydrolysate were also characterized by colorimetric reactions such as Biuret, Ninhydrin, Xanthoproteic, Millon’s, Hopkins-Cole, Sakaguchi, Nitroprusside, Fohl’s, and Amide Tests. All the intact proteins showed a positive result: blue-violet solution for Biuret Test, blue-violet solution for Ninhydrin Test, yellow solution with brown ppt. for Xanthoproteic Test, flesh ppt. for Millon’sTest, pink ring for Hopkins-Cole Test, red-orange solution for SakaguchiTest, red coloration for Nitroprusside Test, black ppt. for Fohl’s Test, red to blue litmus paper and yellow-orange solution for Test for Amides, and red coloration for Pauly Test.

INTRODUCTION Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular of fibrous form, fascilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of adjacent amino residues. The sequence of amino acids in a protein is defined by the sequence of a gene, which is encoded in the genetic code. There are 20 different amino acids found in proteins namely aspartate, glutamate, lysine, arginine, histidine, tyrosine, cysteine, tryptophan, phenylalamine, methionine, serine, threonine, asparagine, glutamine, valine,isoleucine, glycine, alanine, leucine, and proline. There are four points considered in the isolation of an intact protein from its

source. These are the Three-dimensional (3D) structure of the protein (fibrous or globular), interactions that keep the native conformation of the protein, acid-base property, and solubility of protein indifferent solvents. Their solubility can be altered by changing the pH of their environment. Proteins are insoluble and their isoelectric pH, the pH at which the net change is equal to zero. To denature proteins is to disrupt the native conformation. It can be done by subjecting the proteins to extremes of heat and pH, and different denaturing solvents. Denaturing proteins will alter its function, demonstrating a relationship between structure and function. In this experiment, it is intended to isolate myoglobin fro m beef muscle. Myoglobin and hemoglobin are vital to oxygen transport in vertebrates. The former is present in large concentrations in muscles

and is responsible for the red color of the organ. Myoglobin can be isolated by ammonium sulfate precipitation from the buffered muscle extract. Myoglobin is a single-chain globular protein of 153 or 154 amino acids,containing a heme which is an ironcontaining porphyrin prosthetic group in thece nter around which the remaining apoprotein folds. Biologically, active proteins, like myoglobin, are made up of polymers consisting of amino acids linked by covalent peptide bonds. These bonds are broken when the protein undergoes hydrolysis. In hydrolysis, the protein is subjected to extreme conditions usually at high temperatures byprolonged boiling in a strong acid or strong base or using an enzyme such as thepancre atic protease enzyme to stimulate thenaturally occurring hydrolytic process. This will cause denaturation of the protein meaning that the protein’s conformation is altered by the breaking of peptide bonds. This results to a solution containing amino acid fragments which is then called the hydrolysate. Denaturation alters protein function, demonstrating a relationship between structure and function. Hydrolysis of protein and analysis of products are done to obtain information about their compositions. Enzymatic, Acidic and Alkaline Hydrolysis of intact protein was also done in this experiment. The 20 amino acids commonly found as hydrolysis products of proteins contain an alpha-carbonyl, an alphaamino group, and a distinctive R group substituted on the alpha-carbon atom. Hydrolysis of the protein and analysis of the product are done to obtain information about their compositions. Hydrolysis can be carried out by treating the protein with acid, alkali or proteolytic enzymes. In acidic hydrolysis, hydrochloric acid will be used. It vaporizes when heated then comes in

contact with the protein sample and hydrolyzes the sample. Specific reactions are used for the purpose of identifying amino acids and proteins in biological media, for qualitative and quantitative analysis. Biuret test is used to determine peptide bonds. Ninhydrin test is typical for alpha-amino acids. Xanthroproteic test is a test for the detection of aromatic proteins in which concentrated nitric acid reacts with the proteins to form a yellow color that is intensified to orange-yellow by the addition of alkali. Millon’s testis used to demonstrate the presence of the amino acid tyrosine. Hopkins-Cole test is specific for tryptophan group. Sakaguchi test is a test for guanidines, i.e arginine and peptides that contain it. Nitroprusside test is a test for cystinuria. Fohl’s test is used to know if sulfur-containing amino acids are present. Test for amides is used to detect R-groups of asparagines and glutamine. The objectives of this experiment are myoglobin from beef by salt-induced precipitation; and to analyze chemical groups responsible for color reactions and explain the principle involved in each test. MATERIALS AND METHOD A. Isolation of Myoglobin In isolating Myoglobin, 6.0 g of minced beef, (NH4)2SO4 cryztals, 20% buffer diluted (NH4)2SO4 solution with pH 7.5 and centrifuge was required. A 6.0 g minced beef was placed with 6 ml 70 % (NH4)2SO4 solution in a small beaker. The mixture was gently stirred for 1 minute to release the myoglobin. The dark-red extract was expressed into a new beaker using cheesecloth. The extract was centrifuged at 13 000 x g for 15 minutes. A 1.5 ml of supernatant was

transferred into another empty centrifuge tube. A ~0.30-0.35 of (NH4)SO4 crystals ground was added to fine powder then mixed gently until the solid dissolves. The samples were centrifuged again for at least 5 minutes. The supernatant was decanted off and then the appearance of the purified myoglobin residue was described. B. Acid Hydrolysis Myoglobin

of

To hydrolyze the intact protein which is myoglobin, 5 ml of the 6M HCl was added to 0.5 g of the isolated myoglobin in a hard glass test tube. The test tube was properly labeled according to the format given. Then cotton was placed to stopper the tube before it was subjected to autoclaving at 15 psi for 5hours. Alternatively, the protein sample could b eplaced in a sealed container containing 6M HCl. The whole container is then placed in a microwave oven for about 530 minutes with temperatures up to 200oC. This will vaporize the 6M HCl which will come in contact with myoglobin and hydrolyse it. Then, the appearance of the mixture was noted after autoclaving. Then, distilled water with a volume of 10 mL was added and the mixture was transferred into a 250-mL beaker. The mixture was neutralized with 1MNaOH and that neutralized mixture was used for characterization tests and chromatography. C. Qualitative test of Myoglobin and Hydrolysate The intact proteins and hydrolysate were tested with different characterization tests namely: Biuret, Ninhydrin, Xanthoproteic, Millon’s, HopkinsCole, Sakaguchi, Nitroprusside, Fohl’s and Amide. There were 18 test tubes prepared for each of the test reaction. For each test, in a

separate test tubes an inteact protein solution (0.5g of the protein in 1 ml distilled H2O) and 0.5ml of hydrolysate solution was prepared. 1. Biuret Test In the prepared test tube with the intact protein/ hydrolysate solution, 20 drops of 2.5 M NaOH was added and mixed well. Another 2-3 drops of 0.1 M CuSO4 solution was added. The test tube was shaken and the color of the solution was noted. 2. Ninhydrin Test The sample was treated with 6-10 drops of 0.1% ninhydrin solution and was heated in a boiling water bath. The color of the solution was noted. 3. Xanthoproteic Test In the prepared test tube with the intact protein/ hydrolysate solution, 10 drops of concentrated HNO3 was slowly added and was mixed. The color of the solution was noted. Slowly added after was 10 drops of concentrated NaOH and was mixed well and the color of the solution is again noted. 4. Millon’s Test The sample was treated with 5 drops of Millon’s reagent ang the change in colot was noted. 5. Hopkins-Cole Test

For Hopkins-Cole test, 20 drops of Hopkins-Cole reagent as slowly added to the sample protein and was well mixed. The test tube was inclined and 20 drops of concentrated H2SO4 was slowly added along the side. The color and the interface were noted.

NaOH. It was placed in a boiling water bath after. The evolution of gas was tested during heating by placing a moistened red litmus paperover the mouth of the tube. The results were noted. 10. Pauly Test Diazo reagent was prepared by mixing 3-5 drops 1% sulfanilic acid with 3 drops 5% NaNO2 solution. Five drops of the sample and 3-5 drops 10% Na2CO3 was added to the diazo reagent. The appearance of red coloration was noted.

6. Sakaguchi Test The sample was treated with 1 drops of 10% NaOH and 10 drops of 0.02% naphthol solution and was mixed. It was left to stand for 3 minutes, after 3 drops of 2% NaOBr was added and was mixed. The color was noted. 7. Nitroprusside Test In the prepared test tube with the intact protein solution, 0.5 ml of 3 M NaOH was added. Another 0.25 ml of 2%Nitroprussied solution was placed. The decolorization of the solution was noted. 8. Fohl’s Test Five drops of 30% NaOH and 2 drops of 5% (CH3COO)2Pb was added to t. ithe intact protein solution. After which, the test tube was placed in as boiling water bath and the appearance of sediment was noted. 9. Test for Amides Ten drops of the protein sample solution was treated with 1ml of 20%

RESULTS AND DISCUSSION Myoglobin was isolated from the beef by salt-induced precipitation wherein the proteins are less soluble at salt concentrations (high ionic strength)because the salt ions bind most of the water molecules. Myoglobin can be isolated by ammonium sulfate precipitation from the buffered muscle extract. Qualitative Color Reactions of the Intact Proteins

Color Reaction

Biuret Ninhydrin Xantoprotei c

Intact Protein (Myoglobi n) Blue – Violet Sol’n Bule – Violet Sol’n Yellow Sol’n with Brown ppt

Protein Hydrolysa te (Acidic) Light Blue Sol’n Violet Sol’n Pale Yellow Sol’n no ppt.

Millon’s

Flesh Sol’n

No formation of Red Sol’n Hopkins – Pink No Cole Interface formation of Pink interface Sakaguchi Red – No Orange formation Sol’n of Red – Orange Sol’n Nitroprussi Red No Red de Coloration Coloration. Fohl’s Brown ppt. No formation of Brown ppt. Test for Red to Red to Amide Blue Blue Litmus Litmus Paper Paper Pauly Red Red Coloration Coloration

1. Biuret Test (test for peptide bond) The Biuret test is a positive test for proteins but not for amino acid. The evidence for the test consists of formation of a violet-pink complex when cupric ion,in basic solution is added to any polymer such as protein which contains multiple amide bonds. A bluecolored solution indicates a negative test or those fewer than two peptide bonds are present. 2. Ninhydrin Test (test for alpha amino acid) Showed a positive result of blue-violet solution. It is because proteins also contain free amino groups on the alpha-carbon and can react with ninhydrin to produce the blue-violet color.

3. Xantoproteic Test (test for aromatic side chain) The test depends upon a reaction with a specific type of amino acid chain. Aromatic rings often have the ability to undergo nitration reaction, which is the addition of NO2 group to the ring that is why it is a positive test for side chains in tyrosine and tryptophan. With these amino acids present, addition of nitric acid and heat will result in a yellowish-colored solution. Upon addition of base, color will change to orange. Myoglobin is positive of aromatic side chain 4. Millon’s Test (test for phenolic group containing side chain) The color produced in Millon's test is given by derivatives of benzene in which hydrogen in the ring has been replaced by a hydroxyl group. The reaction serves as a test for the presence of tyrosine. 5. Hopkins – Cole Test (test for amino acid tryptophan) The clear violet ring produced is due to the formation of a compound from the glyoxylic acid in the reagent and the tryptophan in the protein. A similar color is produced when sulphuric acid is added to a protein solution in the presence of a trace of formaldehyde. Myoglobin showed a positive result for the presence of tryptophan. 6. Sakaguchi Test (test for the guanidine group of arginine) In basic conditions, alpha naphthol and sodium hypobromite/chlorite react with

the amino acid containing guanidine group to form redorange complexes. Myoglobin sample showed positive result thus, does consist of arginine amino acid. 7. Nitroprusside Test (test for free – SH group) Amino acid with free thiol groups due to cysteine yields a positive result, red to red violet decolorization when introduced to Nitroprusside. Myoglobin yields a positive result for this test. 8. Fohl’s Test (test for sulfur containing amino acid) This reaction is used for determination of S-containing amino acids. Heating of protein solution in an alkaline medium leads to the formation of sulfide (Na2S) if the protein contained sulfur amino acids such as cysteine, cystine or methionine. A positive result is red to red violet decolorization. Further reaction of Na2S will lead acetate a dark brown colored precipitate is formed. 9. Test for Amide Test (test for the presence of asparagine and glutamine) The red litmus paper turned blue indicates a basic component of the gluten, thus gluten is positive for the presence of a basic amino acid. From the data gathered, Myoglobin was chemically determined to be a basic amino acid contain sulfide and peptide bonds ,has an aromatic side chain except for tyrosine. It is also positive for the presence of disulfide bond due to cysteine

10. Pauly Test (test for tyrosine and histidine) Imidazole group reacts with diazotized aulphanilic acid to firm colored azcompounds. In alkaline medium this is red color. Myoglobin yields a positive result for this test.

CONCLUSION All test peroformed on the intact myoglobin yields a positive result. Compared with characterization results of hydrolyzed myoglobin that only Biuret, Ninhydrin, Xantoproteic, Test for amide and Pauly Test provided a positive result. Therefore hydrolysis breaks some components of protein.

REFERENCES Books Pankaja, N. (2011). Essentials of Biochemistry: 2nd edition. (pp 159179) Pavia, D. et al.. (2012). A Microscale Approach to Organic Laboratory Techniques: 5th edition. Websites http://www.thefreedictionary.com/chem ical+analysis http://global.britannica.com/EBchecke d/topic/486122/quantitative-chemicalanalysis http://quizlet.com/8801729/colorreactions-of-proteins-flash-cards/ Kyrk, J. Amino Acids. http://www.johnkyrk.com/aminoacid.swf 1/15/ 2012