Color Reactions of Casein Protein and Basic Hydrolysate

J.C. Garbosa, A.T. Garcia, R.S. Garcia, R.C. Gomez, D.A. Gonong

Group # 4, 2CMT, Faculty of Pharmacy, UST

Abstract

Proteins are probably the most important class of biochemical molecules, although of course lipids and carbohydrates are also essential for life. Casein is a protein that is found in milk which is used independently in many foods as a binding agent. The general objective of this experiment is to isolate the protein Casein from skimmed milk and to analyze the chemical groups responsoble for color reactions and also to explain the principles involved in each test. Biuret Test, Ninhydrin Test, Xanthoproteic Test, Millon’s Test, Hopkins-Cole Test, Sakaguchi Test, Nitroprusside Test, Fohl’s Test and the Test for Amides were all done to a certain portion of the isolated Casein. Basic hydrolysis was done to the other portion of Casein. In addition to this, the different tests performed on the first portion of Casein were also done to the Casein that underwent basic hydrolysis. Various results were obtained and some differences were noted between the color reactions of the isolated Casein and the color reactions of the basic hydrolysates from the isolated Casein.

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

Biuret Test is used for detecting the presence of peptide bonds. It relies on the reduction of copper(II) ions to copper(I), the latter form a complex with the nitrogens of the peptide bonds in an alkaline solution. A violet color indicates the presence of proteins. It is possible to use the biuret reaction to determine the concentration of proteins because (for most proteins) peptide bonds occur with approximately the same frequency per gram of material. The intensity of the color, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to Lambert-Beer's law.

Ninhydrin is a chemical substance mostly used in biochemical laboratories as a reagent for amino acids, which are small molecules that form proteins, as well as in forensics to detect finger prints and faint blood stains, Ninhydrin react with amino acids, producing a colored solution. This protocol is used to detect the presence of amino acids in certain substances by using a solution of alcohol, ninhydrim and water.

Xanthoproteic Test is a test for the detection of proteins in which concentrated nitric acid reacts with the proteins to form a yellow color that is intensified to orange-yellow by the addition of alkali—called also xanthoproteic reaction

Millon's test is given by any compound containing a phenolic hydroxy group. Consequently, any protein containing tyrosine will give a positive test of a pink to dark-red colour. The Millon reagent is a solution of mercuric and mercurous ions in nitric and nitrous acids.

The hopkins-cole test determines the presence of amino acid tryptophan. Some chemists no longer will use the hopkins-cole tst test becasuse it is not completely understood in terms of chemistry. The tryptophan that the hopkins-col e test determines can be defined as an indole nucleus. The Tryptophan creates the violet ring where the two layers meet.

The Sakaguchi Test is used to test for a certain amino acid and proteins. The amino acid that is detected in this test is 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 test is a test wherein sodium cyanide is added first to urine and let stand for approximately 10 minutes. In this time disulfide bonds will be broken by the released cyanide. The destruction of disulfide bonds liberates cysteine from cystine and homocysteine from homocystine. Next sodium nitroprusside is added to the solution and it reacts with the newly freed sulfhydryl groups. The test will turn a red/purple color if the test is positive indicating that there were significant amounts of amino acid in the urine (aminoaciduria).

Fohl’s reaction is used for determination of S-containing amino acids. A black or brown color indicates the presence of the S-containing amino acids.

Bacteria need proteins as their source for essential amino acids. Proteins are large molecules that cannot be brought directly into the bacterial cell. They need to be degraded into their component parts.

Hydrolysis is a chemical reaction during which one or more water molecules are split into

hydrogenand hydroxide ions which may go on to participate in further reactions.

II. Materials and MethodsFor the Alkaline Hydrolysis of Casein, 10 mL of 4M NaOH was added to 0.5 grams of the isolated protein in a hard glass test tube. It was aslo labeled afterwards. The tube was stoppered with cotton and was then submitted to the instructor for autoclaving (15 psi for 5 hours). The appearance of the mixtured after autoclaving was then noted. Ten mL of distilled water was

added. The mixture was then transferred into a 250-mL beaker. The mixture was neutralized using 1M HCl. This neutralized mixture was then used for the characterization tests.

For each of the color reaction test, .5g of the isolated pure Casein protein was mixed with 1mL of distilled water, while .5mL of the hydrolysate was used.

In the Biuret test, 20 drops of 2.5M NaOH was added to the samples and it was mixed well. After this, 2-3 drops of 0.1M CuSO4. The test tube was shaken and the color of the solution was noted.

As for the Ninhydrin test, 6-10 drops of 0.1% Ninhydrin solution was added into the diluted samples. The tube was the heated in a boiling water bath and the appearance of a blue-violet coloration was noted.

Ten drops of concentrated HNO3 was slowly added to the diluted samples. The solution was then mixed and was noted of the color. After this, 10 drops of NaOH was slowly added, mixed and the color of the solutionw as noted.

For the Millon’s test, 5 drops of the reagent was added to the diluted samples and the change in color was noted.

Twenty drops of Hopkins-Cole reagent was slowly added for the Hoplins-Cole test. It was then mixed well. While the test tube was inclined, 20 drops of concentrated H2SO4 was slowly added. The mixture was not shaken and the color of the interface was noted.

As for the Sakaguchi test, 10 drops of 10% NaOH and 10 drops of 0.02% Naphthol solution was added to the samples. It was mixed and let stand for 3 minutes. After 3 minutes, 3 drops of 2% NaOBr was added and the color produced was noted.

For the Nitroprusside test, 0.5mL of 3M NaOH was added to 0.5mL of the sample. After this, 0.25mL of 2% nitroprusside solution was added and the formation of a red solution was noted.

Five drops of 30% NaOH and 2 drops of 5% (CH3COO)2Pb was added to the sample for the Fohl’s test. The test tube was then placed in a water bath. The appearance of a dark(brown or black) sediment was noted.

For the test for Amides, 1 mL of 20% NaOH was added to 10 drops of the sample. The tube was then placed in a water bath. A test for the evolution of gas during heating was done by placing a

moistened red litmus paper over the mouth of the tube. The result was then noted.

III. Results and Discussions

Figure 3.1 isolated casein

Table 3.1 shows the results obtained from the various color reactions

Color Reaction Intact protein (Casein) Basic HydrolysateBiuret purple/ violet solution light blue solutionNinhydrin pale blue-violet solution violet ringXanthoproteic pale yellow solution dark yellow solutionMillon’s white solution pale yellow solutionHopkins-Cole white ring clear yellow solutionSakaguchi colorless solution pale brown solutionNitroprusside yellow solution yellow solutionFohl’s black precipitate, brown solution pale yellow solutionTest for Amides

white basic solution(red->blue litmus paper)

white basic solution(red->blue litmus paper)

The Biuret test for the intact protein was positive indicating the presence of a peptide bond. As for the hydrolysate, the test was positive though the result was not as intense as of the isolated protein’s results.

As for the Ninhydrin test, since a positive result should yield a purple complex, it was negative for the isolated casein and it was positive for the hydrolysate. This indicated the presence of either ammonia or primary or secondary amines.

As for Xanthoproteic test, both had positive results indicating the presence of an aromatic ring but based on the intensity of the yellow color, it was observed that the hydrolysate has more aromatic rings in it.

For Millon’s test, since a brown precipitate or a brick red color is the indicator to confirm the presence of either Tryptophan or Tyrosine, it was negative for both the samples.

Hopkins-Cole test only reacts with proteins containing Tryptophan. Its indicator is the violet cyclic product. Since both the hydrolysate and the casein had no violet color, the test for both of them was negative.

For the Sakaguchi test, a wine red color indicates the presence of Arginine. Since in the casein, only a colorless solution was obtained, it was negetive. But for the hydrolysate, since it had a pale brown color, it is fine to say that it was positive for this test.

The Nitroprusside test only reacts with Cysteine to yield a red complex. Since for both the samples, it turned to yellow solutions, the test was negative to either.

As for the Fohl’s test, it was positive for the Casein indicating that it has an S-containing amino acid. It turned out to be negative for the hydrolysate.

For both the 2 samples, it was positive in the test for Amides because of the initially red litmus paper which was placed over the mouth of the tube turned to blue.

As for the conclusion, the casein and the basic hydrolysate had different reactions for Biuret test, Ninhydrin test, Sakaguchi test, while it had the same result in Xanthoproteic test, Millon’s test, Hopkins-Cole test, Nitroprusside test, Fohl’s test and the test for Amides.

IV. References

http://www.elmhurst.edu/~chm/vchembook/565proteins.html

http://www.wisegeek.com/what-is-casein.htm

http://www.worldlingo.com/ma/enwiki/en/

http://www.ehow.com

http://www.chemistry.mcmaster.ca/~chem2o6/labmanual/expt11/2o6exp11.html

http://www.reference.com/motif/Science/

http://www.cerlabs.com/experiments/10875404480.pdf