QUALITATIVE COLOR REACTIONS OF INTACT AND ACID HYDROLYZED PROTEIN CASEIN

Bettina Maria Q. Acua, Enrico Joseph C. Aguinaldo, Alyanna Patrice L. Arceo, Jasmine Grace C. Asinas and Sigrid Jessa R. Bello Group 1 2G Pharmacy Biochemistry Laboratory

ABSTRACT
Chemical analysis can either be qualitative or quantitative. In qualitative analysis we only identify the substances present in a given sample. We are not anymore concerned with the quantity of each substance. On the other hand, in quantitative analysis, we are concerned with determining the amount of each component present in a sample. Our objective for this experiment is to isolate the protein casein from skimmed milk and to analyze the chemical groups responsible for the color reactions and also to explain the principles involved in each test. Biuret Test, Ninhydrin Test, Xanthoproteic Test, Millons Test, Hopkins-Cole Test, Sakaguchi Test, Nitroprusside Test, Fohls Test, the Test for Amides and Pauly Test were all done to the first portion of the isolated casein and also to the other part which underwent acidic hydrolysis. Various results were obtained and some differences were noted between the color reactions of the intact casein and the color reactions of the acidic hydrolysates from the isolated casein. The Biuret test is used to detect the presence of peptide bonds. It should produce a blue-violet coloration, an indication a positive result. The Ninhydrin test is a test used for an alpha-amino acid. Amino acids contain free amino group and free carboxylic acid group that reacts together with Ninhydrin and produces a blue-violet solution The Xanthoproteic test detects the side chains of aromatic amino acids which will produce a yellow solution. The Millons test is given by any compound containing a phenolic hydroxy group. This test determines tyrosine residues. A red precipitate or red solution indicates a positive result.

INTRODUCTION
Amino acids have a variety of chemically reactive groups. The reactions for side chains, a-amino, and acarboxyl groups can be used to characterize both free amino acids and proteins.

Figure1. General structure of an amino acid

Proteins are probably the most important class of biochemical molecules, although of course lipids and carbohydrates are also essential for life. Proteins are the basis for the major structural components of animal and human tissue. Proteins are natural polymer molecules consisting of amino acid units. The number of amino acids in proteins may range from two to several thousands. Casein is a protein that is found in milk and used independently in many foods as a binding agent. It is part of a group called phosphoproteins, a collection of proteins bound to something containing phosphoric acid. Casein is a salt, meaning it has no net ionic charge, of the element calcium. It is not susceptible to denaturing because of its structure. Our group was assigned to isolate casein from skim milk through isoelectric precipitation with acetic acid. After precipitating casein, several samples were used for the qualitative color reactions. And after which we did the acidic hydrolysis of the isolated casein for Biuret test, Ninhydrin test, Xanthoproteic test, Millons test, Hopkins-Cole test, Sakaguchi test, Nitroprusside test, Fohls test, Test for Amide and Pauly test.

The Hopkins-Cole test is specific only for tryptophan. It should produce a purple ring coloration at the interface indicating a positive result. The Sakaguchi test is a test for guanidines, i.e arginine and peptides that contain it; in alkaline solution they give a red coloration, which contains anaphthol and sodium hypochlorite. The Nitroprusside test is used to determine the presence of cystein, the only amino acid containing a sulfhydryl group (-SH). A red solution should be observed. The Fohls test is used to detect presence of sulfur containing amino acid. A black or brown solution should be observed indicating a positive result. The test for Amides is used to detect asparagines and glutamine. A red limus paper should turn to blue to indicate a positive result. In the Pauly test, it involves the diazotization of sulphinicilic acid in the presence of sodium nitrite and sodium carbonate. This test answers for tyrosine and histidine residues. It should produce a red solution.

EXPERIMENTAL
A. Samples used
Intact Casein, Hydrolyzed Casein

10. Pauly Test

The diazo reagent was first prepared by mixing 3-5 drops of 1% sulfanilic acid with 3 drops of 5% NaNO2 soulution. Then, 5 drops of the sample and 3-5 drops of 10% Na2Co3 was added to the diazo reagent. The appearance of a red coloration was noted.

B. Procedure 1. Biuret Test

In the prepared test tube with the intact protein solution, 20 drops of 2.5 M NaOH was added and mixed well. Another 2-3 drops of 0.1 M CuSO4 solution was added. The test tube was shaken and the color of the solution was noted.

RESULTS AND DISCUSSION

The intact protein casein and acidic hydrolysate were both tested to identify and determine the functional groups that they contain.

2. Ninhydrin Test
The sample was treated with 6-10 drops of 0.1%ninhydrin solution and was heated in a boiling water bath. The color of the solution was noted.

Table 1. Qualitative Color Reaction Results of Intact and Hydrolyzed Casein

Test Biuret Test Ninhydrin Test Xanthoproteic Test Millons Test Hopkins-Cole Test Sakaguchi Test Nitroprusside Test Fohls Test Color Reaction of Intact Protein Purple solution (+) Blue-Violet Solution (+) Yellow Solution (+) Flesh (+) Purple ring at Interface (+) White precipitate (-) Yellow solution (-) Black/ Brown Solution (+) Red --> Blue Litmus Paper (+) Red solution (+) Color Reaction of Acid Hydrolysate Greenish Brown Solution (-) Orange-Brown Solution (-) Yellow Orange Solution (+) Yellow Solution (-) Clear Brown Solution (-) Light Brown Solution (-) Orange-Brown Solution with Precipitate (-) Amorphous Black precipitate (Clear Solution) (+) Red --> Blue Litmus Paper (+) Yellow Solution with Black precipitate (-)

3. Xanthoproteic Test
In the prepared test tube with the intact protein solution, 10 drops of concentrated HNO3 was slowly added and was mixed. The color of the solution was noted. Slowly added after was 10 drops of concentrated NaOH and was mixed well and the color of the solution is again noted.

4. Millons Test
The sample was treated with 5 drops of Millons reagent and the change in color was noted.

5. Hopkins-Cole Test
20 drops of Hopkins-Cole reagent was added to the samples. The test tube was inclined and 20 drops of concentrated H2SO4 was slowly added along the side. The color and the interface were noted.

6. Sakaguchi Test
For this test, 10 drops of 10% NaOH and 10 drops of 0.02% naphthol solution was added to the samples. It was mixed well and left to stand for 3 minutes. Test for Amide Pauly Test

7. Nitroprusside Test
In the prepared test tube with the intact proteinsolution, 0.5 ml of 3 M NaOH was added. Another 0.25 ml of 2% Nitroprusside solution was placed.The decolorization of the solution was noted.

8. Fohls Test
Five drops of 30% NaOH and 2 drops of 5%(CH3COO)2Pb was added to the intact protein solution. After which, the test tube was placed in a boiling water bath and the appearance of sediment was noted.

9. Test for Amides

Ten drops of the protein sample solution was treated with 1mL of 20% NaOH. It was then placed in a boiling water bath. The evolution of gas was tested during heating by placing a moistened red litmus paper over the mouth of the tube. The results were noted.

From the obtained data, the intact casein protein showed positive results in the Biuret test, Ninhydrin test, Xanthoproteic test, Millons test, Hopkins-Cole test, Fohls test, test for Amide, and in the Pauly Test. We can say that this intact casein protein contains peptide bonds (Biuret test), a-amino acids (Ninhydrin test), side chains of aromatic amino acids (Xanthoproteic test), Tyrosine residues (Millons test ), Tryptophan residues (Hopkins-Cole test), R-groups of asparagine and glutamic acid (test for Amide), sulfurcontaining amino acids (Fohls test), histidine and tyrosine (Pauly test). On the other hand, the acid hydrolyzed casein protein yielded positive results on the Xanthoproteic test, Fohls test and test for Amide. This determines that the acid hydrolysate contains side chains of aromatic amino acids (Xanthoproteic test), sulfur-

containing amino acids (Fohls test) and R-groups of asparagine and glutamic acid (test for Amide).

REFERENCES
From books Boyer, R. (2006). Concepts in Biochemistry 3rd edition. Hoboken NJ: John Wiley & Sons Inc. pp.63-90 From the internet http://ebookbrowse.com/gdoc.php?id=313192321&url =018da670785040500c6acf370a6a3fdc 12/28/12 http://www.graduatestudiesus.com/2010/10/modernexperimental-biochemistry-3d-ed_08.html 12/28/12 http://bookmoving.com/book/modern-experimentalbiochemistry_94134.html 1/6/13