Characterization of Intact Casein and Acid Hydrolyzate through Color Reactions Reyes, Kendrick Don, Rostrata, Myrr Kea*,

Roxas, Josemarie Emmanuel, Susi, Lindley Group 9, 3BIO6 Department of Biological Science, University of Santo Tomas, Manila, Philippines Abstract
Proteins are amino acids linked by peptide bonds and Acid hydrolyzate has free amino acids due to the breaking of peptide bonds during the hydrolysis. Proteins and hydroyzates are characterized using color reactions. Biuret test, Sakaguchi test, Ninhydrin test, Xanthoproteic test, and Hopkins-Cole test were used to characterize the intact protein and acid hydrolyzate. Intact protein has positive results in the entire test except in Ninhydrin test. The Acid Hydrolyzate has positive results in Ninhydrin and Xanthoproteic tests.

Introduction Proteins are polymers of amino acids that are linked by peptide bonds (Pratt &, Cornley, 2011). Peptide bonds break during hydrolysis. Aside from the breaking of peptide bonds, destruction of tryptophan, serine, and threonine, and oxidation of methionine also occur during acid hydrolysis (http://www.nihs.go.jp/dbcb/Bio-Topic/amino.pdf). The protein used in this experiment is casein. Casein is the main protein in milk; it is a phosphoprotein and it is soluble in dilute acid and alkali (Shankara, 2008). Proteins can be characterize through various color reactions like Biuret test, Sakaguchi test, Ninhydrin test, Xanthoproteic test, and Hopkins-Cole test. Proteins with one or more peptide bonds will have positive result in Biuret test; the CuSO4 reacts with the peptide bond that yields to violet complex (Shankara, 2008). Sakaguchi test produce pink or red color when naphthol reacts with guanidine derivatives (Baker, 1947). It is for the detection if arginine is present (Patil & Muskan, 2009). Ninhydrin test is for the detection of free amino groups that has a visible result of deep purple solution (Peller, 1998). The reaction of the ninhydrin with the

amino acid is an oxidative deamination (Bezkorovainy & Rafelson, Jr., 1996). Xanthoproteic test is for the detection of aromatic group in amino acids or proteins; the aromatic ring undergoes nitration reaction that yields nitro derivatives which is yellow in acidic conditions; this yellow solution will turn orange under alkaline condition (Patil & Muskan, 2009). Hopkins-Cole test is used to test for the presence of tryptophan; it is also called glyoxylic reaction (Patil & Muskan, 2009). The mentioned tests are significant for qualitative detection of protein and amino acids (Shankara, 2008). (C.W.Pratt & Cornely, 2011) This experiment aims to characterize the intact protein and acid hydrolyzate using various chemical tests. Methodology The dried casein was cut into small pieces and was placed in a mortar. Thirty milliliters of distilled water was added. The casein was grinded until it becomes a fine protein suspension. The protein suspension and the acid hydrolyzate (from experiment 1) were tested by various color reactions. In the Biuret test, three drops of protein suspension were put in the spot plate and three drops of acid hydrolyzate was put on an adjacent well. One drop of 2.5M of NaOH was added on each well. Both are mixed well. A drop of 0.01M of CuSO4 solution was added in each well and was mixed well. The colors produced were noted. In the Sakaguchi test, five drops of protein suspension were put in the spot plate and five drops of acid hydrolyzate was put in the adjacent well. A drop of 10% NaOH and a drop of 0.02% naphthol solution were added in each well and were mixed well. After about 3 minutes, a drop of 2% NaOBr was added in each well. The colors produced were noted. In Ninhydrin test, ten drops of protein suspension were put in a small test tube and ten drops of acid hydrolyzate was added in another small test tube. One milliliter of water was added in each test tube. Ten drops of 0.1% ninhydrin solution were added in each test tube and was mixed well. Both test tubes were heated in a boiling water bath for 2-3 minutes. The colors produced were noted. In Xanthoproteic test, ten drops of protein suspension were put in a small test tube and ten drops of acid hydrolyzate were put in another test tube. One milliliter of water was added in each test tube. Three drops of concentrated HNO3 were slowly added and was mixed well. The colors produced were noted. Both test tubes were heat in a boiling water bath for one minute. The solutions were cooled with flowing water. Concentrated NaOH was added drop by drop

until the solution is alkaline; the solution was tested with litmus paper. The colors produced were noted. In Hopkins-Cole test, two drops of protein suspension were added in a small test tube and two drops of acid hydrolyzate were added in another test tube. Two milliliter (40 drops) of Hopkins-Cole reagent was added in each test tube and was mixed well. The test tubes were inclined and two mL concentrated H2SO4 was slowly added down the side of the test tube until two layers form. The test tubes were not disturbed and the color of the interphase was noted.

Results and Discussion Biuret test will produce a violet-pink solution, an indication of positive result, if a compound has two or more peptide bond. Negative result of biuret test will produce a bluecolored solution. The intact protein has a positive result which means there are more than one peptide bonds present in the compound while the acid hydrolyzate has a negative result because the peptide bond were broken during hydrolysis. Sakaguchi test has a positive result to those compounds with arginine; red color solution is the result of the reaction of the guanido of the free arginine or arginine residues with naphthol and NaOBr (Shankara, 2008). Intact Protein yields red solution in Sakaguchi test, it means it has a positive result and it has an arginine residue. The Acid hydrolyzate has a negative result. Ninhydrin test, if positive will results to violet solution due to the reaction of free amino groups with the ninhydrin. The - amino acid that reacts with ninhydrin will yield to CO2, ammonia, and hydratin and the hydratin and ammonia will react with the ninhydrin that will yield to Ruhemanns purple (Shankara, 2008). Intact protein has a negative result, it means it doesnt have a free amino group while the acid hydrolyzate has a positive result; this is because during the acid hydrolysis, the peptide bonds break resulting to free amino acids. Acid hydrolyzate has free amino acids. In Xanthoproteic test, the yellow solution before heating is a positive result; the yellow solution is the result of the nitration reaction in the aromatic group. Intact protein and acid hydrolyzate has an aromatic group. Positive result in Hopkins-Cole test is indicated by purple interphase; it is due to the condensation of tryptophan with aldehyde in acidic condition (Patil & Muskan, 2009). The intact protein is positive in this test therefore tryptophan is present in the protein. The acid hydrolyzate is negative in this test because the tryptophan was destroyed during the acid hydrolysis. Summary of the visible results of the color reactions are shown in table 1.

Table 1: Result of intact protein and acid hydrolyzate in various tests. Results Test Biuret Test Sakaguchi Test Ninhydrin Test Xanthoproteic Test: Before heating After heating Hopkins-Cole Test Intact Protein Purple solution Red solution Turbid white solution Colorless solution with white precipitate Yellowish solution with white precipitate Purple is the color of the interphase Acid Hydrolyzate Gray solution Yellow orange solution Blue violet solution Yellowish solution Light yellow solution Light yellow solution

Conclusion The casein intact protein has more than one peptide bond, an arginine residue, an aromatic group, and tryptophan. The acid hydrolyzate has free amino groups and aromatic group but does not have more than one peptide bond, an arginine group, and tryptophan.

References
(n.d.). Retrieved July 9, 2012, from http://www.nihs.go.jp/dbcb/Bio-Topic/amino.pdf Baker, J. R. (1947). The Histochemical Recognition of Certain Guanidine Derivatives. Quarterly Journal of Microscopical Science , 115-121. Bezkorovainy, A., & Rafelson, J. M. (1996). Concise Biochemistry. New York: Marcel Dekker, Inc. C.W.Pratt, & Cornely, K. (2011). Essential Biochemistry 2nd ed. United States: John Wiley & Sons, Inc. J.R.Peller. (1998). Exploring Chemistry: Laboratory Experiments in General, Organic, & Biological Chemitry. New Jersey: Prentice Hall. Patil, U. K., & Muskan, K. (2009). Essentials of Biotechnology. New Delhi: I.K International Publishing House Pvt. Ltd. Shankara, S. (2008). Laboratory Manual for Practical Biochemistry. New Delhi: Jaypee Brothers Medical Publishers.