Characterization of Protein using Qualitative Color Reactions of Intact and Alkaline Hydrolyzed Gluten

Gamaliel P. Galigao, Jessica V. Guanlao , Zarah Pauline Jimenez, Maolenn Lavadia and Kamille Angeline Lim Group 5 2-D Pharmacy, Faculty of Pharmacy, University of Santo Tomas

Abstract Protein hydrolysis breaks down proteins to their constituent amino acids and peptides. Alkaline hydrolysis is considered as complete hydrolysis because all peptide bonds are attacked therefore complete break down into component parts could be obtained. The objectives are to perform alkaline hydrolysis on the isolated gluten, to enumerate the advantages and disadvantages of alkaline hydrolysis, to analyze chemical groups responsible for color reactions and to explain the principle involved in each test. The isolated gluten was treated with 4M NaOH and autoclaved for five hours. Intact gluten and basic hydrolysates were used in performing Biuret test, Ninhydrin test, Xanthoproteic test, Millons test, Hopkins-Cole test, Sakaguchi test, Nitroprusside test, Fohls test, test for amides an d Pauly test. The intact protein was negative for Hopkins-Cole only and the basic hydrolysate had negative results in Biuret test, Sakaguchi test, and Nitroprusside test. In conclusion, gluten is chemically determined to be a basic amino acid containing sulfide bonds, peptide bonds and aromatic side chain except tyrosine. It is also positive for the presence of disulfide bond due to cysteine.

Introduction Protein hydrolysis breaks down proteins to their constituent amino acids and peptides. Alkaline or basic hydrolysis is one method of protein hydrolysis which utilizes a strong base as chemical catalyst. Alkaline hydrolysis is considered as complete hydrolysis because all peptide bonds are attacked therefore complete break down into component parts could be obtained. One of the principal advantages of basic hydrolysis compared to acid hydrolysis is the retaining of tryptophan; on the other hand, racemization occurs in basic hydrolysis and threonine, serine, cysteine and cystine are destroyed. In the process of basic hydrolysis, arginine is converted to ornithine and urea. The final result of protein hydrolysis is the amino acid that makes up the protein. The 20 amino acid commonly found as hydrolysis product of protein contain alpha carboxyl group, an alpha amino group and a distinctive

R-group substituted on an alpha carbon atom. Protein hydrolysis is done to obtain information about the composition of the protein. Characterization of protein involves performing of qualitative color reactions in order to determine the proteins composition. Reagents are used to react with intact and hydrolyzed protein samples for different kinds of qualitative tests. The Biuret test is used to detect the presence of peptide bonds while the Ninhydrin reaction is a typical test for an alpha amino acid. The Xanthoproteic test detects side chains of aromatic amino acids while the Millons and Hopkins-Cole tests determine tyrosine and tryptophan residues, respectively. The Nitroprusside test is used to find out if sulphur-containing amino acids are present; test for amides is used to detect R-groups of asparagines and glutamine.

Gluten is the rubbery mass that remains when wheat dough is washed to remove granules and water-soluble constituents. Gluten is a mixture of proteins not readily soluble in water that occurs in wheat and most other cereal grains. Gluten is present in flour which makes the production of leavened baked goods possible because the chain-like gluten molecules form elastic networks that traps carbon dioxide and expands with the gluten. Materials and Methods A. Alkaline Hydrolysis of Intact Gluten The experiment utilized 0.5 gram isolated gluten, hard glass, cotton as stopper and autoclave. Ten millilitres of 4M NaOH was added to 0.5g isolated protein in a hard glass test tube. The tube was plugged with cotton and autoclaved (15 psi for 5 hours). The appearance of the hydrolysate was noted after autoclaving. The hydrolysate filtered into a 250-ml beaker. The filtrate was neutralized with 1M HCl. B. Qualitative Color Reactions The samples were prepared for each test in separate test tubes an intact protein solution (0.5g of gluten in 1ml distilled water) and 0.5ml of basic hydrolyzed sample. For the Biuret test, 20 drops of 2.5M NaOH was added to the samples and mixed. Another 3 drops of 0.1M CuSo4 solution was added. The test tube was shaken and the color of the solution was noted.

For Ninhydrin test the samples were treated with 10 drops of 0.1% ninhydrin solution and were heated in a boiling water bath. The color of the solution was noted. In Xanthoproteic test, 10 drops of concentrated HNO3 was added to the samples and was mixed. The color of the solution was noted. Ten drops of concentrated NaOH was slowly added and mixed. The color of the solution was noted again. In Millons test, 5 drops of millons reagent was added to the samples and the change in color was noted. In Hopkins-Cole test, 20 drops of Hopkins-Cole reagent was slowly added to the samples and was mixed. The test tubes were inclined and 20 drops of concentrated H2SO4 was slowly added along the side. The color at the interface was noted. For the Sakaguchi test, 10 drops of 10% NaOH and 10 drops of 0.02% naphthol solution were added to the samples and were mixed. The tubes were left to stand for 3 minutes. Three drops of 2% NaOBr and mixed. The color produced was noted. For Nitroprusside test, 0.5 ml of 3M NaOH was added to 0.5 the sample. Then, 0.25 ml of 2% Nitroprusside solution was added and the color of the solution formed was noted. In Fohls test, 5 drops of 30% NaOH and 2 drops of 5% (CH3COO)2Pb were added to the samples. The tubes were placed in a boiling water bath. The

appearance noted.

of

dark

sediment

was

specific for proteins, it is necessary to apply several tests. Table 1 Result of Qualitative Color Reactions of Intact Gluten and Basic Hydrolysate of Gluten
Test Biuret Ninhydrin Xanthoproteic Intact Gluten Blue violet solution Blue violet solution Yellow solution Peach solution Clear ring Red solution Dark brown precipitate Red solution Red to blue litmus paper Red solution Basic Hydrolysate Grey solution Dark violet solution Yellow solution Peach solution Violet ring Light brown solution Dark brown precipitate Black precipitate Red to blue litmus paper Red solution

In the test for amides, 1ml of 20% NaOH was added to 10 drops of the sample. The tubes were placed in a boiling water bath. To test the evolution of gas, a moistened red litmus paper was placed over the mouth of the tube during heating and the result was noted. To prepare the diazo reagent for the Pauly test, 5 drops of 1% sulfanilic acid was mixed with 3 drops of 5% NaNO2 solution. After preparing the reagent, 5 drops of the sample and 5 drops of 10% Na2CO3 was added to the diazo reagent. The appearance of a red coloration was noted. Results and Discussion A. Alkaline Hydrolysis of Intact Gluten The intact gluten was hydrolyzed in order to destroy peptide bonds to form free amino acids. After autoclaving, the hydrolysate appeared to be a turbid solution with suspended thin films. The hydrolysate was filtered in order to eliminate the unnecessary suspended thin films. B. Qualitative Color Reactions A number of qualitative color reactions have been devised which are useful for the detection of proteins. Owing to the complexity of the protein molecule and the difficulty of obtaining a single pure protein, these test were used with the knowledge that they test for the specific chemical groupings on the protein structure, or more generally for the groupings which most proteins contain. Since no test is absolutely

Millons Hopkins-Cole Sakaguchi Fohls

Nitroprusside Test for Amides Pauly

Presence of peptide bonds is detected by performing a chemical test named Biuret test. The Biuret Reagent is made of sodium hydroxide and copper sulfate. The blue reagent turns violet in the presence of proteins, and changes to pink when combined with short-chain polypeptides. Complexation of cupric ion is the principle involved in this test. Triketohydrindene hydrate, commonly known as Ninhydrin reacts with amino acids to form a purple colored imino derivative. Ninhydrin is a powerful oxidizing agent which leads to the oxidative deamination of alphaamino groups. In xanthoproteic test, boiling concentrated nitric acid reacts with

tyrosine, tryptophan and phenylananine to yield yellow products. This reaction involves the nitration of benzene nucleus in alkaline medium. Amino acids that contain aromatic nucleus undergo this reaction. Millons test is for compounds containing a phenolic hydroxy group and involves the principle complexation of phenolic compound. Millons reagent is mercuric ion in acid. A positive result for Millons test is a peach colored solution. In Hopkins-Cole test, glyoxilic acid reacts with the indole ring of tryptophan to yield a violet ring at the junction. The principle involved is complexation of charged transfer. Sakaguchi test is for the presence of guanidinium group. Complexation is the principle involved and a positive result yields a red solution. The presence of sulfur containing amino acids (cysteine and methionine) is determined using the nitroprusside test. In Nitroprusside test, sodium nitroprusside reacts with sulfur to produce a red solution. Ionic precipitation of sulfur is the principle involved in this test. Fohls test detects the presence of cysteine and methionine which gives a positive result of dark brown or black precipitate. Test for amides is used to find out if asparagine and glutamine are present. A moistened red litmus paper was placed over the mouth of the test tubes to detect evolution of gas during

heating. A positive result turns the red litmus paper to blue. Pauly test detects the presence of histidine and tyrosine. The diazo reagent reacts with tyrosine or histidine and produces a red orange solution. The intact gluten gave a negative result for Hopkins-Cole test because it does not contain tryptophan. The basic hydrolysate gave a negative result for the Biuret test because the peptide bonds have been broken down. A negative result was given by the Sakaguchi test because arginine was converted to ornithine and urea. Nitroprusside test also gave a negative result because cyteine was already destroyed. In conclusion, Gluten is chemically determined to be a basic amino acid containing sulfide and peptide bonds. Gluten also has an aromatic side chain except tyrosine. It is also positive for the presence of disulfide bond due to cysteine. References Crisostomo, A., et al. Laboratory Manual in General Biochemistry; Quezon City: C & E Publishing, Inc., 2010 Bernas, G. et al. Basic Laboratory Studies in Biochemistry; University of Santo Tomas Publishing House; 1994 Bailey, J. Ollis, D., Biochemical Engineering Fundamentals Second Edition; McGraw-Hill, Inc., 1986

Toporek, M. Basic Chemistry of Life; C. V. Mosby Company, 1971 Date Retrieved December 28,2012 from http://home.earthlink.net/~dwyerg /HL%20Labs/proteins%20and%2 0amino%20acids.htm