ISOLATION AND CHARACTERIZATION OF PROTEINS (ALKALINE HYDROLYSIS OF CASEIN) Mary Catherine Sarte, John Emmanuel Sy, Allurie Umel,

Franklin Yap, Mary Christine You Abstract

Casein is the main protein found in the milk of mammals including cows, goats, and humans. Casein is the predominant phosphoprotein ( S1, S2, , ) 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. Subjected to qualitative color reactions and paper chromatography had been done to analyze the different amino acid components of the protien

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. Amino acids have a great variety of chemical reactive groups providing a wide range of reactivity proteins. The reactions for individual side-chain radicals and -amino and -carboxyl groups are specific both for free amino acids and proteins, whereas the reaction for peptide group is characteristic of the protein and peptides. Specific reactions are used for the purpose of identifying amino acids and proteins in biological media, for qualitative and quantitative analysis. Biuret reaction which accounts for determining peptide bonds, Ninhydrin reaction is typical for amino acids. Xanthroproteic test detect sidechains of aromatic amino acids and Millons and Hopkins- Cole tests are for the determination of tyrosine and tryptophan residues, respectively. Fohls test is used to know if sulfur-containing amino acids are present and sulfosalicylic acid for the imidazole ring of histidine residues. The hydrolysis of amides under acidic condition is basically the same as protein hydrolysis, because peptide bond link is the same as an amide link. If you scale this up to a polypeptide chain, each of the peptide links will be broken in exactly the same way. That means it will end up in a mixture of the amino acids that constitute the protein. Protein hydrolysates can be tested qualitatively by different reagents and as well as paper chromatography. Partition paper chromatography is widely employed for the determination and separation of amino acids. The solvent migrates along a strip of paper and carries amino acid dissolved in it.

Experimental
A. Alkaline Hydrolysis of Intact Protein Casein extracted from skimmed milk was hydrolyzed by adding 10ml of 4M NaOH to 0.5g isolated protein in a hard glass test tube for autoclaving(15psi for 5 hours) after that 10 ml of distilled water was added and the mixture was neutralized with 1M HCL after it was tested with different qualitative color tests. The sample was filtered and the filtrate was collected for characterization test and paper chromatography. B. Qualitative Color Reaction The casein hydrolysate was tested with different characterization reagents namely: Biuret, Ninhydrin, Xanthroproteic, Millon,Hopkins-Cole, Sakaguchi, Fohl, and Diazo tests. Eight test tubes were prepared for each of the test reaction. Each test tube consisted of 1 mL of distilled water added to 0.5 mL of hydrolyzed samples. Biurets test In Biuret test 20 drops of 2.5 NaOH and 2-3 drops of 0.1 M CuSO4were mixed and shaken observed for color change, turned to blue violet. Ninhydrin test For Ninhydrin test, 6-10 drops of 0.1 % Ninhydrin solution was added to the dilute sample and heated in a water bath. The appearance of a blue-violet coloration was taken note of. Xanthoproteic test In Xanthroproteic test, concentrated nitric acid and concentrated sodium hydroxide 10 drops each were added slowly and then mixed. Color changes after each addition were observed turned to light yellow. Millons test Five drops of Millons reagent was added to the diluted sample with the appearance of a peach color taken note of. Hopkins-Cole test For Hopkins-Cole test, 20 drops of HopkinsCole reagent was slowly added to the sample and mixed well. Sulfuric acid about 20 drops was slowly added alongside the inclined test tube. The color of the interface was white and taken note of.

Sakaguchi test After the addition of 10 drops of Sakaguchi reagent and mixed, it was let stand for 3 minutes and 3 drops of 2% NaBOr was added. A yellow color was indicated and taken note of. Nitropusside test For Nitropusside,0.5ml of 3M NaOH and 0.25 ml 2% nitropusside solutionwere added. A formation of red color was taken note of. Fohls test In Fohls test, 5 drops 30% NaOH and 2 drops 5% (CH3COO)2Pb. Placed the test tube in a boiling water bath. Note the appearance of a dark( black-brown) sediment. Test for Amides Adding 1ml of 20% NaOH to 10 drops of the sample. Heat in a boiling water bath a moistened red litmus paper was placed at the mouth of the test tube to test for evolution of gas. The litmus paper turned red to blue and was taken note of.

Results and Discussions

The protein casein exists as a colloidal suspension in milk and gives milk its characteristic white, opaque appearance. If the casein in the milk is hydrolyzed into peptides and amino acids, it will lose its opaqueness. Qualitative Color Test for Hydrolyzed Casein In Table.1 the characterization of the hydrolyzed protein, data results of intact protein of the same tests were compared with the former to show the difference of the reactions in characterization. Table 1. Results of Qualitative Color Reaction of Intact and Hydrolyzed Proteins

Biuret test showed positive results because of the presence ofa copper(II) ion forms a violet-colored complex in an alkaline solution. In Ninhydrin When that nitrogen is deprotected, a ninhydrin test yields blue which is positive. Xanthoproteic and Millons test yielded positive because of the presence of tyrosine residue. In Hopkins-Cole test showed postivie at the presence of the animo acid tryptophan that appears a violet solution. The Sakaguchi test showed a positive yellow at the presence of arginine. Fohls test is positive due to the presence of cysteine having s black-brown sediment. The alkaline hydrolysis of amides actually involves reaction with hydroxide ions, but the result is similar enough that it is still classed as hydrolysis. Refferences
Campbell, Mary; Farell Shawn. (2008). Biochemistry (6th ed.). Canada: Brooks/Crole. The Biochemistry Department (2008). Laboratory Manual in General Biochemistry.Manila : University of Santo Tomas

http://en.wikibooks.org/wiki/Biochemistry http://chemistry2.csudh.edu/rpendarvis/carboxder.htm l#struct http://www.ncbi.nlm.nih.gov/pmc/articles/PMC47762/

Color reaction Biuret Ninhydrin Xanthoproteic Millon s Hopkins-Cole Sakaguchi Nitroprusside Fohl s

Intact protein Light-blue Blue violet Yellow Peach Purple Orage-yellow Red Black-brown sediment

Protein Hydrolysate Blue-violet Blue-violet Light yellow Peach White Yellow Red Brown-black

Test Amides

for Red to blue Blue to red litmus litmus paper paper