Color Reactions of Intact Protein and Enzymatic Hydrolysate

R.F Vila, K.Y. Visco, R.G. Vivar, J.C. Zafra, J.C. Ziganay

Abstract: Proteins are one of the essential groups of biochemical molecules, along with the carbohydrates and lipids. Casein is a protein that is commonly found in mammalian milk which is used independently in some food as a binding agent. The experiment aims to isolate the protein casein from skimmed milk and to study the biochemical groups accountable for color reactions and give explanation to the principles involved in each test. Biuret Test, Ninhydrin Test, Xanthoproteic Test, Millons Test, Hopkins-Cole Test, Sakaguchi Test, Nitroprusside Test, Fohls Test and the Test for Amides were all done to a certain portion of the isolated casein. Enzymatic hydrolysis was done to the other segment of the protein. In addition to this, the different tests performed on the first portion of casein were also done to the casein that was being experimented for enzymatic hydrolysis. Various results were obtained and some differences were noted between the color reactions of the isolated Casein and the color reactions of the enzymatic hydrolysates

Introduction: Amino acids have a variety of chemically reactive groups. Biuret Test is used for identifying the presence of peptide bonds. It relies on the reduction of copper(II) ions to copper(I), the latter form a complex with the nitrogens of the peptide bonds in an alkaline solution. A violet color indicates the presence of proteins. The intensity of the color, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to Lambert-Beer's law. Ninhydrin is a chemical substance generally used in biochemical laboratories as a reagent for amino acids, which are minute molecules that form proteins, as well as in forensics to detect finger prints and faint blood stains, Ninhydrin react with amino acids, producing a colored solution. This protocol is used to detect the presence of amino acids in certain substances. Xanthoproteic Test is a check for the recognition of 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 test is given by any compound containing a phenolic hydroxy group. As a result, any protein containing tyrosine will give a positive test of a pink to brick red color. The Hopkins-Cole test verifies the presence of amino acid tryptophan. The tryptophan that is determined can be defined as an indole nucleus. The Tryptophan creates the violet ring where the two layers assemble. Sakaguchi Test is used to test for a certain amino acid and proteins. The amino acid that is detected in this test is arginine. A positive result yields a bright red color in the presence of arginine. Nitroprusside test is a test wherein sodium cyanide is added first to urine and letting it stand for about 10 minutes. As a result, disulfide bonds will be broken by the released cyanide. The destruction of disulfide bonds liberates cysteine from cystine and homocysteine from homocystine. Next sodium nitroprusside is

added to the solution and it reacts with the newly freed sulfhydryl groups. The test will turn to a red/yellow color if the test is positive indicating that there were considerable amounts of thiol groups (Cys, Met) in the urine. Fohls reaction is used for the detection of S-containing amino acids. A black or brown sediment indicates the presence of the S-containing amino acids (Cys, Met). Test for Amides detects the presence of basic amino acids (Asp, Glu) and gives a red to blue change in litmus paper. Pauly test detects the presence of histidine and tyrosine and shows a red coloration. Enzymatic hydrolysis is the catalytic decomposition of a chemical compound by reaction with water, such as the conversion of cellulosic substances into fermentable sugars by the addition of specific enzymes.

mixed with 1mL of distilled water, while .5mL of the hydrolysate was used. In the Biuret test, 20 drops of 2.5M NaOH was added to the samples and mixed well. After this, 2-3 drops of 0.1M CuSO4. The test tube was shaken and the color of the solution was noted. As for the Ninhydrin test, 6-10 drops of 0.1% Ninhydrin solution was added into the diluted samples. The tube was heated in a boiling water bath and the appearance of a blue-violet coloration was noted. Ten drops of concentrated HNO3 was slowly added to the diluted samples. The solution was then mixed and was noted of the color. After this, 10 drops of NaOH was slowly added, mixed and the color of the solution was noted. For the Millons test, 5 drops of the Millons reagent was added to the diluted samples and the change in color was noted. Twenty drops of Hopkins-Cole reagent was slowly added to the samples and they were mixed well. While the test tube was inclined, 20 drops of concentrated H2SO4 was slowly added. The mixture was not shaken and the color of the interface was noted. As for the Sakaguchi test, 10 drops of 10% NaOH and 10 drops of 0.02% Naphthol solution was added to the samples. It was mixed and let to stand for 3 minutes. Three drops of 2% NaOBr was added and the color produced was noted. For the Nitroprusside test, 0.5mL of 3M NaOH was added to 0.5mL of the sample. After this, 0.25mL of 2%

Methodology:
For the Enzymatic Hydrolysis of the intact protein, 1g/100ml of distilled water protein mixture was prepared. Ten ml of protein mixture and 10 ml of saturated protease solution was mixed. Alternatively, 0.050 g of protease may be added directly to 50 ml of protein mixture. Ten ml of 0.1 M phosphate buffer, pH 7.5 was added to the protein mixture. For 60 minutes, the tube was incubated in a water bath for about 3540C. The mixture is cooled before using it in the procedures for color reactions. For each of the color reaction test, .5g of the isolated pure Casein protein was

nitroprusside solution was added and the formation of a red solution was noted. For Fohls test, 5 drops of 30% NaOH and 2 drops of 5% (CH3COO)2Pb was added to the sample for the Fohls test. The test tube was then placed in a water bath. The appearance of dark (brown or black) sediment was noted. For the test for Amides, 1 mL of 20% NaOH was added to 10 drops of the sample. The tube was then placed in a

water bath. A test for the evolution of gas during heating was done by placing a moistened red litmus paper over the mouth of the tube. The result was then noted. Lastly, for Paulys test, the diazo reagent was prepare by mixing 3-5 drops of 1% sulfanilic acid with 3 drops of 5% NaNO2 solution. Five drops of the sample and 3-5 drops of 10% Na2CO3 were added to the diazo reagent. A positive result will yield a red coloration.

Results and Discussions Table 3.1 shows the results obtained from the various color reactions

Color Reaction Biuret Ninhydrin Xanthoproteic Millon s Hopkins-Cole Sakaguchi Nitroprusside Fohl s Test for Amides Pauly s Test

Intact protein Blue-violet coloration Deep blue/ purple coloration Red-orange solution Brick red color violet ring Bright red solution Red to yellow coloration Gray/black sediment Red-blue litmus paper Red coloration

Enzymatic Hydrolysate Brown solution with green precipitate Deep purple coloration Reddish-brown color Colorless solution with orange precipitate Violet ring No reaction Red to orange coloration Brown sediment red-blue litmus paper Reddish brown solution
hydrolysate because of the amino acid present due to the breaking of peptide bonds to shorter fragments. As for Xanthoproteic test, redorange solution was indicated for the intact protein as well as for the hydrolysate which even though it gave a reddish brown coloration.

The Biuret test for the intact protein was positive indicating the presence of a peptide bond. As for the hydrolysate, the test was different because in enzymatic hydrolysis, bonds are broken through peptidases and proteolytic enzymes. As for the Ninhydrin test, a positive result was shown because of the deep purple coloration and same with the

For Millons test, brick red color was observed on the intact protein indicating the presence of tyrosine. However, the hydrolysate didnt yield a positive result which meant the absence of the amino acid tyrosine. Hopkins-Cole test only reacts with proteins containing Tryptophan. Its indicator is the violet cyclic product. The intact protein and the hydrolysate showed that they both have a tryptophan amino acid. For the Sakaguchi test, a bright red color indicates the presence of Arginine. The hydrolysate proved that there is no presence of arginine because it has no reaction. The Nitroprusside test only reacts with Cysteine to yield a red to yellow coloration. The hydrolysate yield a red to orange coloration which means that there is a presence of cysteine. As for the Fohls test, the hydrolysate compared to the intact protein gave a result of brown sediment which means that there is presence of Scontaining amino acid. The intact protein showed that the basic amino acid turned the red litmus to blue litmus paper. Also, the hydrolysate turned the red litmus to blue due to the presence of basic amino acid. As for Paulys test, intact proteins red coloration has the almost the same result with the hydrolysate which is reddishbrown coloration indicating the presence of histidine or tyrosine. As a conclusion, the enzymatic hydrolysate showed that it contained some amino acids like histidine, cysteine,

tryptophan, etc. because it has the same or almost the same results compared to the intact protein. The test designated a negative result for the hydrolysate whenever there was an absence of the particular amino acid being tested like for the Millons test that has no amino acid tyrosine.

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