CASEIN PROTEIN ISOLATION AND CHARACTERIZATION OF ENZYMATIC HYDROLYSATE BY PAPER CHROMATOGRAPHY AND QUALITATIVE COLOR

REACTIONS 2DPH Group 3 Constantino, Faye; Dagcuta, Arnoika; *De Alva, Mycaela; Decena, Marck Paulo; Enriquez, Floen Michael ABSTRACT Casein is the main protein found in the milk of mammals including cows, goats, and humans. Casein is the predominant phosphoprotein ( 1, S2, , k) that accounts for nearly 80 percent of proteins in cow milk and cheese. Milk-clotting proteases act on the soluble portion of the caseins, K-Casein, thus originating an unstable micellar state that results in clot formation. It is recognized for its excellent amino acid profile,slow digestion, and interesting peptides (casomorphins, casokinins, casoxins, etc). In this experiment, Casein was isolated, hydrolyzed and neutralized from non-fat powdered milk. Casein was first isolated by adding 10% Acetic acid until if formed an amorphous mass. The isolated casein was further hydrolyzed and neutralized for the characterization. A positive result from the enzymatic hydrolysate was observed under Biuret, Ninhydrin, Xanthoproteic, Hopkins-Cole, Nitropusside, Fohls, Test for amides and Pauly Tests. In the paper chromatography, serine has the lowest Rf value with 0.07 along with Arginine with a Rf value of 0.08. Tyrosine has the highest Rf value with 0.24. I. 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. Several amino acids combine to form peptide bonds. Proteins contain polypeptide units (several peptide units). When a protein is hydrolyzed, it breaks down into smaller units (tri and dipeptides), eventually forming amino acids. Specific reactions are used for the purpose of identifying amino acids and proteins in biological media, for

qualitative and quantitative analysis. The biuret test is used to detect the presence of peptide bonds while the Ninhydrin reaction is a typical test for an -amino acid. Xanthoproteic test detects side chains of aromatic amino acidswhile the Millons and Hopkins-Cole tests determine tyrosine and tryptophan residues, respectively. The Nitroprusside test is used to find out if sulfur-containing amino acids are present; test for amides is used to detect R-groups of asparagine and glutamine. Test for amides is used to detect R-groups of asparagine and glutamine. The objective of the experiment is to determine the amino acid components of casein, which can be done by partition paper chromatography, which is widely employed for the separation of amino acids. The solvent migrates along a

strip of paper and carries amino acid dissolved in it II. Methodology a. Paper Chromatography A 12cm x 15cm filter paper was used to facilitate paper chromatography. A margin of 1.0 cm was lined from the top and bottom to the filter paper. Thirteen equidistant points were marked to indicate where the selected ten amino acids and the three hydrolyzed proteins were to be spotted. The ten amino acids were spotted twice using a capillary tube and the hydrolysates were spotted five times. The filter paper was then stapled to become a cylinder. A 1000mL beaker and a watch glass were prepared to serve as the developing chamber. The solvent system used was a mixture of 1-Butanol, acetic acid and water in 4:1:5 ratio and it was poured into the beaker and left undisturbed inside the developing chamber. Then the cylindered filter paper was placed inside the beaker, covered with watch glass and allowed to ascend uninterrupted for almost an hour. The solvent front was marked with a pencil and allowed to dry. The chromatogram was sprayed with a 1% Ninhydrin reagent and heated on top of a hot plate for some minutes. Purple and yellow spots were observed and encircled. Rf values were computed using the following formula: ! = Rf or retention factor is the ration between the distance traveld by the solute and the distance traveled by the solvent. It is used to identify the unknown by comparing its Rf value to a Rf value of known compounds.

b. Qualitative Color Reactions The reactions for side chains, -amino and -carboxyl groups can be used to characterized both free amino acids and proteins. For each colorimetric test, separate test tubes of intact protein solution (0.5g of the protein in the 1 mL distilled water) and 0.5 mL of hydrolyzed sample were used. The Biuret Test, Ninhydrin Test, Xanthoproteic Test, Millons Test, Hopkins-Cole Test, Sakaguchi test, Nitropusside test, Fohls test, Test for Amides, and Pauly Test were used to characterize the free amino acids and proteins. Biuret Test: 20 drops of 2.5 M NaOH was added to a test tube containing the intact protein and another 20 drops were added to the test tube containing the enzymatic hydrolysate. Then to each test tube, 2-3 drops of 0.1M CuSO solution were added. Both test tubes were shaken and the color was noted. Ninhydrin Test: In each test tube, 6-10 drops of 0.1%ninhydrin solution were added into the intact protein and enzymatic hydrolysate. Both test tubes were then heated in a boiling water bath. Xanthoproteic Test: Ten drops of concentrated HNO3 solution was slowly added to the diluted samples and were mixed. Then,10 drops of concentrated NaOH was added and the color was noted. Millons Test: To each of the diluted samples, 5 drops of Millons reagent were added

while noting the change in color. Hopkins-Cole Test: To the diluted samples, 20 drops of Hopkins-Cole reagent was slowly added and mixed well. The test tubes were then inclined the test tube and 20 drops of concentrated H2SO4 was added along the side. The change in color was then noted. Sakaguchi Test: To each of the diluted samples, 10 drops of 10%NaOH and 10 drops of 0.02%naphthol solution was added and the test tubes were then left untouched for 3 minutes. Nitroprusside Test: 0.5 mL of 3M NaOH was added to the diluted samples. Then, 0.25 mLof 2% of nitroprusside solution was added. Fohls test: Five drops of 30%NaOH and 2 drops of 5%(CH3COO)2Pb was added to the diluted samples. Both test tubes were then places in a boiling water bath and the change in color was observed. Test for Amides: To each of the diluted samples, 1 mL of 20%NaOH were added and both test tubes were placed in a boiling water bath. While in the water bath, a red litmus paper was held at the opening of each test tube to test for the evolution of gas. Pauly Test: First, the diazoreagent was prepared by mixing 3-5 drops of 1% sulfanilic acid with 3 drops of 5% NaNO2 solution. Then, 5 drops of the sample and 3-5

drops of 10%Na2CO3 were added to the diazoreagent. A red coloration was noted. III. Results a. Paper Chromatography Table 1. Rf values of each amino acids

Amino Acid Standards Enzymatic Protein Hydrolysate Distance travelled (cm) Solvent front (cm) Rf Value Tryptophan 1.89 9.0 0.21 Arginine 0.72 9.0 0.08 Proline 1.44 9.0 0.16 Cysteine 2.07 9.0 0.23 Serine 0.63 9.0 0.07 Aspartic Acid 2.16 9.0 0.24 Tyrosine 3.78 9.0 0.42 Histidine 0.9 9.0 0.1 Glycine 1.8 9.0 0.2 Alanine 2.16 9.0 0.24 The table 1 shows the results of the Separation and Identification of amino acids by Paper Chromatography. Serine has the lowest Rf value with 0.07. Followed with Arginine with a Rf value of 0.08. Tyrosine has the highest Rf value with 0.24. Table 2. Qualitative Color Reactions Color Reaction Enzymatic Protein Hydrolysate Biuret + (Purple solution) Ninhydrin + (Purple solution) Xanthoproteic + (Yellow solution) Millons - (White Turbid solution) Hopkins-Cole + (Pink interface) Sakaguchi - (Clear solution) Nitropusside + (Yellow solution) Fohls + (Clear solution)

Test for Amides + (Clear solution) Pauly + (Red orange solution) Table 2 shows the results of qualitative color reactions of enzymatic hydrolysate. A positive result was observed under Biuret, Ninhydrin, Xanthoproteic, Hopkins-Cole, Nitropusside, Fohls, Test for amides and Pauly Tests. IV. Discussion a. Paper chromatography Paper chromatography is the separation and migration of the amino acids based on its affinities to the stationary and mobile phases. Different factors affect the affinity of a substance and these are the following: polarity, pH, molecular weight, structure, and shape of the molecule. Based on the Table 1, amino acids serine and arginine have the lowest Rf value. Components with smaller Rf value travel more slowly, a polar compound, bond to the cellulose of the paper more quickly and have a higher affinity to the stationary phase. On the other hand, tyrosine moved farthest from the point of the origin. Consequently, it has the greatest Rf value. It means that tyrosine has the greatest affinity toward the mobile phase. The Rf values of each component indicated the polarities of the amino acids. A higher Rf value denotes lower polarity while a lower Rf value implies that the component is polar. b. Qualitative Color Reactions The Biuret test is a test done for the presence of peptide bonds. It is a positive test for proteins but not for amino acids. The evidence for a

peptide bond is the formation of a violet-pink solution. The formation of a violet-pink solution is due to when the cupric ion, in a basic solution is added to any polymer such as proteins, which contains multiple amide bonds. The enzymatic hydrolysate shows positive result for peptide bonds. The Ninhydrin test is a test for the presence of amino acid. The positive result of amino acid is detected by the yielding of a purple solution. The enzymatic hydrolysate showed negative results. Xanthoprotheic test is used for amino acids containing aromatic groups that are derivatives of benzene such as tyrosine and tryptophan. These aromatic groups can undergo reactions that are characteristics of benzene and benzene derivatives. One such reaction is the nitration of a benzene ring with nitric acid. The amino acids that have activated benzene ring can readily undergo nitration. This nitration reaction, in the presence of activated benzene ring, forms yellow product. The enzymatic hydrolysate on the other hand, showed some presence of aromatic groups. Millons test is specific for the detection of tyrosine. Tyrosine is the only amino acid that contains a phenol group on which a hydroxyl group is attached. A positive result will yield a red precipitate. The enzymatic hydrolysate showed a negative result. Hopkins-Cole test is specific for the detection of the presence of tryptophan. The color 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 sulfuric acid is added to a protein solution in the presence of a trace of formaldehyde. The reaction is used as a test for formaldehyde in milk. The formation of a purple solution indicates a positive result. The enzymatic hydrolysate yielded a positive result. Sakaguchi test is used to test the only amino acid,which contains a guanidine group which is arginine. Arginine gives a red color with -naphthol, in the presence of an oxidizing agent like Bromine solution. The enzymatic hydrolysate showed negative result for the presence of arginine. Nitroprusside Test is specific for the detection of cystein, the only amino acid containing sulfhydryl group. This group reacts with nitroprusside in the presence of excess ammonia. Positive results show a red complex. However, the enzymatic hydrolysate showed positive results, yielding a yellow solution. Fohls test is performed for the determination of S- containing amino acids. The solutions were heated for the formation of sulfide. If the test yielded a positive result, the solution shows a red solution or a further reaction of NA2S will lead to a dark brown precipitate. The enzymatic hydrolysate yielded a positive result. Test for Amide is done for the presence of Asparagine and Glutamine. When the red litmus paper turned blue, it indicates a basic component of the myoglobin and enzymatic hydrolysate. Pauly test is specific for histidine and tyrosine wherein it deals

with the formation of azo dyes. Its positive result is the formation of red solution. The enzymatic hydrolysate yielded a positive result. V. References Crisosotomo, A. et.al Laboratory Manual in General Biochemistry Murray RF, Harper HW, Granner DK, Mayes PA, Rodwell VW. (2006).Harper'sIllustrated Biochemistry. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC47762/