Color Reactions of Protein Casein

Abstract

In this experiment different kinds of processes were done to isolate the different kinds of proteins. The protein casein was extracted from skimmed milk through isoelectric precipitation, albumin was also extracted from skimmed milk through heat denaturation, gluten was isolated from wheat flour through difference in solubility, and myoglobin from beef meat through salting out or salt-induced precipitation. The main objective of the qualitative color reaction of the proteins is to analyze the chemical groups present in the protein and explain the principle involved in each test. Biuret Test, Ninhydrin Test, Xanthoproteic Test, Millon’s Test, Hopkins-Cole Test, Sakaguchi Test, Nitroprusside Test, Fohl’s Test, Test for Amides, and Pauly Test were the different tests that were performed. Each test corresponds to the different reagents that were used. In the different tests performed, positive and negative results are gathered that indicates the presence or absence of a particular amino acid in the protein.

Introduction

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 thousand.[3]Biologically active proteins are polymers consisting of amino acids linked by covalent peptide bonds.[1]The properties of a protein are determined by theorder or sequence of the amino acids its molecule, and by the three-dimensional structure of the molecularchain. The chain folds and twists andthen forming its conformational structure which gives its distinctive properties.[2] The structure of an amino acid contains an amino group, a carboxyl group, and a R group which is usually carbon based and gives the amino acid its specific properties. These properties determine the interactions between atoms and molecules, which are: van der Waals force between temporary dipoles, ionic interactions between charged groups, and attractions between polar groups. [3] Amino acids have a variety of chemically reactive groups. The reactions for side chains, α-amino, and α-carboxyl groups can be used to characterize both free amino acids and proteins. [4] The objective of the qualitative color reaction is to analyse and verify the presence of the different amino acids in the protein casein. The tests involved in the experiment are: Biuret test, Ninhydrin test, Xanthoproteic test, Millon’s test, Hopkin’s-Cole test, Sakaguchi test, Nitroprusside test, Fohl’s test, Test for Amides, and Pauly test. Biuret test is used to determine the presence of peptide bonds which uses the active component: sodium hydroxide and copper sulfate. The principle of this reaction is reduction. A positive result for Biuret test will yield a violet or blue-violet solution. Ninhydrin test is a test for a free α-amino group. The positive visible result is a blue-violet solution. The principle in this reaction is decarboxylation and then complexation. The Xanthoproteic test is used to determine the presence of an aromatic ring. 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.[5] Millon’s test is specific to phenol containing structures which is the tyrosine because it is the only common phenolic amino acid. Millon’s reagent is concentrated HNO3, in which mercury is dissolved. As a result of the reaction a red precipitate or a red solution is considered as positive test. [5] Hopkins-Cole test is specific for the amino acid, tryptophan which involves the principle of hydrolization and the positive result is the formation of a violet ring. The amino acid that is detected in the Sakaguchi test is arginine. Since arginine has a guanidine group in itsside chain, it gives a red color with α-naphthol in the presence of an oxidizing agent like bromine solution. [5] The Nitroprusside test is specific for cysteine, the only amino acid containing sulfhydryl group (-SH). This group reacts with nitroprusside in the presence of excess ammonia. [5] Fohl’s test has the specificity to S-containing amino acids and yields a black or brown precipitate for its positive result. The active component in this test is sodium hydroxide and lead (2+) diacetate. The test for amide uses 20%NaOH as its active components and has a specificity on glutamine and asparagine amino acids. A positive result of this test is when a red litmus paper which will turn into blue. This indicates that a basic component, which is the amide, is present. The principle involved in this test is heating. Pauly test determines the presence of tryptophan, tyrosine and hisitidine through diazotization with the reagents 1% sodium nitrite and 10% sodium carbonate. The positive visible result is red coloration of the solution.

Methodology

After the obtainment of casein from skimmed milk an intact protein solution was prepared by adding 0.5g of protein in 1mL distilled water. The solution was distributed in ten different test tubes and 0.5mL of hydrolyzed sample was added in each. The first test that was done is the Biuret test. 20 drops of 2.5M NaOH was added to the sample and was mixed well. The next step done was to add 2-3 drops of 0.1M of CuSO4. The test tube was shaken and the color of the solution was noted. [4] The second test is the Ninhydrin test. 10 drops of 0.1% ninhydrin solution was added in the diluted sample and was heated in a boiling water bath. The color of the solution was observed. [4] The third test done was Xanthoproteic test. Ten drops of concentrated HNO3 was slowly added to the sample then was mixed. After the change in color was noted ten drops of concentrated NaOH was slowly added and the change in color was then observed. [4] Millon’s test was done by adding 5 drops of Millon’s reagent to the sample and the color change was noted. [4] In Hopkins-Cole test, 20 drops of Hopkins-Cole reagent was added slowly to the sample and was mixed well. The test tube was then inclined and 20 drops of concentrated H2SO4 were added slowly. It was ensured that the mixture was not shaken nor mixed in order to observe an accurate result. [4] In the Sakaguchi test, 10 drops of 10%NaOH and 10 drops of 0.02% naphthol solutiom were slowly added to the sample. It was then mixed and left for three minutes to stand. Three drops of 2% NaOBr was added to the mixture and was mixed. [4] Nitroprusside test was done by adding 0.5mL of 3M NaOH to 0.5mL of the sample. Then, 0.25mL of 2% nitroprusside solution was added. [4] In Fohl’s test, 5 drops of 30% NaOH and two drops of 5% (CH3COO)2Pb was added to the sample and was placed in a boiling water bath. The appearance of dark sediment was noted. [4] Test for amides was done by the addition of 1mL 20% NaOH to ten drops of the sample and was placed in a boiling water bath. A moistened red litmus paper was placed above the mouth of the test tube to test the acidity or basicity of the gas from the mixture. [4] The last test was the Pauly test. A diazo reagent was prepared by mixing 3-5 drops of 1% sulfanilic acid with three drops 5% NaNO2 solution. Five drops of sample and 3-5 drops of 10% Na2CO3 was added to the diazo reagent.[4]

Results

The following results were gathered and are presented in the following table:

Table 1. Color Reactions and Results Color Reaction | Intact Protein | Biuret | Violet solution | Ninhydrin | Turbid white solution | Xanthoproteic | Yellow solution with precipitate | Millon’s | White precipitate | Hopkins-Cole | Colorless solution w/ violet interference | Sakaguchi | colorless solution | Nitroprusside | Yellow solution | Fohl’s | Brown solution w/ precipitate | Test for Amide | Red litmus paper to Blue litmus paper; brown solution | Pauly | Red solution |

Based on the table presented, different results were obtained according to their different specificity. In the Biuret test, a violet solution was obtained which indicates that peptide bonds are present that represents the presence of amino acids. The second test, Ninhydrin test, gave a turbid white solution which indicates a negative result which means that a free α-amino group is absent. In Xanthoproteic test, an yellow solution with precipitate was obtained that specifies the presence of an aromatic ring. A positive visible result in Millon’s test is a red precipitate or solution. In the experiment, a white precipitate was observed which designates that the test is negative to the presence of a phenolic group or tyrosine. In Hopkin’s-Cole test, a violet interference was formed which indicates the presence of tryptophan. A colorless solution was obtained in the Sakaguchi test which specifies the absence of the amino acid, arginine. Casein showed a negative result in the Nitroprusside test since it yielded a yellow solution which does not indicate the presence of cysteine. The visible positive result for Nitroprusside is the change of color into a red solution or a red complex. Fohl’s test for casein yielded a brown precipitate which is a positive result which indicated the presence of a S-containing amino acid. The test for amide in casein resulted into a positive outcome. The gas that has evolved from the solution turned the red litmus paper into a blue litmus paper. This outcome indicates the presence of glutamine or asparagine. The final test performed is the Pauly test which yielded a red solution. The red solution indicates a positive result for typtophan, tyrosine, and histidine.

Discussion

Table 1 gives us a better understanding on the composition of the different amino acids and the polypeptide bonds connecting the protein casein.

References

[1]
Campbell, M. and Farrell, S. (2012). Biochemistry (7th edition). Canada: Brooks/Cole.

[2]
Proteins. Retrieved January 7, 2012, from http://encyclopedia.farlex.com/Protein+(biochemistry).

[3]
Ophardt, Charles E. (2003). Virtual Chembook El mhurst College
[4]
Crisostomo, A., et al (2010). Laboratory Manual in General Biochemistry. Philippines: C&E Publishing, Inc.
[5]
Qualitative Analysis of Amino Acids and Proteins.Retrieved January 7, 2012 from http://www.chem.boun.edu.tr/webpages/courses/Chem415/Chem%20415%20Experiment%202.pdf.