Isolation and Characterization by Color Reaction of the Protein Casein

*B. Ocampo, F. Padilla, K. M. Park, R. Payuyo, D. Ponciano, J. V. Regala

Group 7, 2CMT, Faculty of Pharmacy, UST

Abstract

The objective is to isolate it in order to perform hydrolysis to break its components and color reaction for its characterization. Acetic acid was added to isolate casein by the formation of a solid particle called curd. Then, to breakdown its amino acid components acid hydrolysis was performed by using 6M HCl. Color reaction was performed to characterize the protein and its functional groups plus the free amino acids. This white solid particle that precipitated after the addition of acetic acid is confirmed to be casein.

Introduction

Protein--Casein

Protein is a biological molecule which can be classified into two: Fibrous and Globular. Fibrous protein is a long chain or sheet of amino acids while Globular protein is a compact kind of spheroidal shape. They are naturally occurring polymer composed of amino acid as its monomer. Each monomer is connected by peptide bonds. Peptide bonds are amide bonds formed between the carboxylic acid group of one amino acid and the carboxylic group of a second amino acid. The solubility of amino acids is largely dependent on the solution pH. The structural changes in amino acid take place at different pH values that alter the relative solubility of the molecule. In acidic conditions, both amino and carboxylic groups are protonated. In basic groups, both groups are unprotonated. The aim of this experiment is to isolate the protein--Casein, an example of protein found in milk, by adding acetic acid and to identify what amino acids were present in casein.

Casein (Isolation and Color Reaction)

Casein is phosphoprotein which has phosphate groups attached to the hydroxyl groups of some of the amino acids side-chains. It exists in milk as a calcium salt, calcium caseinate.  It has an isoelectric point of pH 4.6. This means it is insoluble in solutions with a pH less than 4.6. The pH of milk is 6.6; therefore, casein has a negative charge at this pH and is solubilized as a salt. When hydrochloric acid was added, the casein was neutralized due to the addition of protons to the phosphate group and the pH is decreased to that of the isoelectric point (point at which the net charge is zero and the casein is insoluble). Because of this, casein became polar and caused its precipitation.

Acetic acid, an example of weak acid, was added to isolate casein from milk. Moreover, certain functional groups in amino acids and proteins can react to produce characteristically colored products. The color intensity of the product formed by a particular group varies among proteins in proportion to the number of reacting functional or free groups present and their accessibility to

the reagent. The following tests were performed to characterize the protein and determine the presence of what amino acid:

Biuret Test was used to detect the presence of peptide bonds. It should produce a violet color.

Ninhydrin Test was used for an alpha-amino acid. Amino acids contain free amino group and free carboxylic acid group that react together with Ninhydrin to produce a blue-violet solution.

Xanthoproteic Test detects the side chains of aromatic amino acids which will produce yellow solution. Xanthoproteic comes from the greek word which means “yellow”. Hence the name of the test.

Millon’s test determines tyrosine residues. A red precipitate or red solution indicates a positive result.

Hopkins-Cole Test is specific for tryptophan. It should produce a violet solution. Sakaguchi test is to detect arginine. The sample to be tested is treated with alpha-

naphthol and sodium hypochlorite. A positive result yields a reddish wine color when arginine is present. 

Nitroprusside is used to determine the presence of cystein, the only amino acid containing a sulfhydryl group (-SH). A red solution should be observed.

Fohl’s test is used to detect presence of sulfur containing amino acid. A black or brown solution is a positive result.

Test for Amides is used to detect asparagines and glutamine. Yellow solution should be produced.

Materials & Methods

Isolation

To be able to isolate casein from milk, the latter’s pH should be decreased at a point where casein is insoluble. And that is 4.6. To adjust the pH of milk, 10% acetic acid was added dropwise to the solution while it is being heated on a 40 degree Celsius temperature. If it happens that the temperature went beyond the required, casein will disintegrate. When the desired pH was reached, curds formed and the milk was almost opaque. This only shows that casein, the protein that gives the milk its milky color, is being separated from it.

Qualitative Color Reaction

Biuret Test :Twenty drops of 2.5M NaOH then 2-3 drops of 0.1 M CuSO4 solution.

Everything was mixed into one consistency.

Ninhydrin Test:

Six to ten drops of 0.1% ninhydrin solution to the diluted sample and heated it to a water bath. The colore produced was noted.

Xanthoproteic Test: Ten drops of concentrated HNO3 was slowly added then the color was noted.

After that, 10 drops of concentrated NaOH was added and again another color was produced.

Millon’s test: Five drops of Millon’s reagent was placed and a color was produced.

Hopkins-Cole Test:Twenty drops of Hopkins-Cole reagent was added to the sample. The test tube

was inclined and 20 drops of concentrated H2SO4 wad added. But this time, the mixture was not shaken.

Sakaguchi test: Ten drops of 10% NaOH and 10 drops of 0.02% naphthol solution was added and

was let to stand for 5 minutes. Three drops of NaOBr was added afterwards. The colore was noted.

Nitroprusside: Three molar of NaOH, 0.05 ml, was added to 0.5ml sample. Then 0.25 ml of 2%

nitroprusside was added and red solution should be expected to form.

Fohl’s test: Five drops of 30% NaOH and 2 drops 5% (CH3COO)2Pb was added then it was

placed on a boiling water bath. Dark brown sediment should be observed.

Test for Amides: One milliliter of 20% NaOH was added to 10 drops of the sample. It was placed

in a boiling water bath. Moistened litmus paper was placed on top of the test tube. The result was noted.

Results & Discussion

The expected result from this experiment is a white solid particle which will precipitate from milk when acetic acid was added. The procedure did produce a white precipitate. The milk is positive to have casein. It can be concluded that the negativity of casein was neutralized by the addition of 10% acetic acid. And since the isoelectric point of casein is 4.6, the point at which it is insoluble, it precipitated when the solution reached this pH.

In this experiment, various color-producing reagents were used to qualitatively detect the presence of certain functional groups in casein.

Table 1: Results of the Color reaction

Color Reaction ResultBiuret Violet solution

NinhydrinBlue violet deposit and solution

Xanthoproteic Yellow solutionMillon’s Fleshy solutionHopkins-Cole Violet solutionSakaguchi Red solutionNitroprusside Yellow solutionFohl’s Black and brown sediments

Test for AmideBasic(red litmus paper to blue); yellow solution

The results show that all tests were positive and that all the amino acid that these tests determine are present:

Biuret confirmed the presence of peptide bonds which produced a violet color. Ninhydrin gave a positive result for an alpha-amino acid which gave out a blue-violet color. Xanthoproteic confirmed the presence of aromatic amino acid by producing a yellow solution. Millon’s test didn’t exactly produce a red solution so tryrosine might not be present. Hopkins-Cole showed that tryptophan is present when it produced a violet solution. Sakaguchi test yielded a red solution which is a positive result for the presence of arginine. Nitroprusside is negative for it yielded a yellow solution. Fohl’s test was positive with the black brown sediment. The test for amide confirmed the presence of aspargine and glutamine when it produced a yellow solution in a water bath.

To conclude, a white solid precipitate signified that milk has casein and that it is composed of several amino acids joined by a peptide bond. By using color reaction, the following kinds of amino acids were found to be present: aromatic amino acid, tryptophan, arginine, sulfur-containing amino acid, aspargine, and glutamine.

Reference

Book:

Crisostomo, A. C. (2010). Laboratory Manual in General Biochemistry (p.18). Quezon City: C&E Publishing, Inc.

Website:

http://homepages.ius.edu/DSPURLOC/c122/casein.htm http://www.cerlabs.com/experiments/10875404480.pdfhttp://www.reference.com/motif/Science/sakaguchi-test-amino-acid-proteins