Qualitative Color Reaction for Casein

M.A. Llado; *A.T.N Lopez; S.R. Maningas; R.J. Martin; P.C. Medina

Abstract:
Our group was assigned to isolate the protein Casein from skim milk through isoelectric precipitation with acetic acid. After precipitating casein, several samples were used for the qualitative color reactions. After which we did the Alkaline Hydrolysis of Intact Protein for Buiret test, Ninhydrin test, Xanthoproteic test, Millon s test, Hopkins-Cole test, Sakaguchi test, Nitroprusside test, Fohl s test, Test for Amide and Pauly test. The intact protein was negative in Millon s Test and Hopkins-Cole Test. However it showed a positive result only for Fohl s Test. The alkaline or basic hydrolysate had negative results in Biuret test, Ninhydrin test, Millon s test, Sakaguchi test, and Nitroprusside test.

of the element calcium. It is not susceptible to 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 (1). The objectives of this experiment are to isolate the following proteins like Casein from skimmed milk by isoelectric precipitation. To analyze chemical groups responsible for color reaction and explain the principle involved in each test. To perform acid, alkaline and enzymatic hydrolysis on the isolated proteins and enumerate the advantages and disadvantages of each type of hydrolysis (2). 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, collections of proteins bound to something containing phosphoric acid. Casein is a salt, meaning it has no net ionic charge, A. Qualitative Color Reactions of Casein 1. Biuret Test In the prepared test tube with the intact protein solution, 20 drops of 2.5 M NaOH was added and Methodology In isoelectric precipitation, the isoelectric point must be achieved wherein the net charge of the protein will be equal to zero. It is done by precipitating a complex mixture until the protein is precipitated at a certain pH level (5). denaturing because of its structure (3). Isolation of a protein is a method for isolating a single type of protein from a complex mixture. The importance of isolating proteins is to characterize their solubility, acid-base property, function, structure, and interactions. Proteins can be separated depending on their size, shape, charge, hydrophobicity and physiochemical

properties. Some of the methods that are commonly used are isoelectric precipitation, heat denaturation,

solubilization, salt-induced precipitation, chromatography, 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.

A 0.5 ml of NaOH and 10 drops of 0.02% naphthol solution was added to the sample. Mixed then for 3 minutes it was let stand. Three drops of 2% NaOBr was added and mixed. The formation of red

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

solution was noted.

8. Fohl s Test Five drops of 30% NaOH and 2 drops of 5% (CH3COO)2 Pb to the sample. After which the tube

3. Xanthoproteic Test For this test the sample was slowly added with 10 drops conc. HNO3 and mixed then the color of the solution was noted. After which the sample was slowly added 10 drops conc. NaOH. It was mixed and the color of the solution was noted.

was placed in a boiling water bath. The appearance of dark (black or brown) sediment was noted.

9. Test for Amides A 1 ml of 20% NaOH to 10 drops of the samples was put in the sample. The tube was placed in a boiling water bath. The evolution of gas was tested during

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

heating by placing a moistened red litmus paper over the mouth of the tube. The results were noted.

10. Pauly Test 5. Hopkins-Cole Test In the prepared test the sample was slowly added with 20 drops Hopkins-Cole reagent and mixed well. The test tube was inclined and 20 drops conc. H2SO4 was slowly along the side. The color and interface was noted. B. Alkalyne Hydrolysis of Casein 6. Sakaguchi test The sample was treated with 10 drops of 10% NaOH and 10 drops of 0.02% naphthol solution and was mixed. It was left to stand for 3 minutes, after, 3 drops of 2% NaOBr was added and was mixed. The color produced was noted. The intact protein which is casein was hydrolyzed by adding 10 ml of 4 M NaOH to 0.5 g isolated protein and was placed in a hard glass test tube and was labeled. The tube was covered with cotton which will act as a stopper and was submitted for autoclaving in 15psi for 5 hours. The appearance was noted and 10 ml of 7. Nitroprusside Test distilled water was added and mixed well. The mixture was placed into a 250ml beaker. The mixture was The diazo reagent was prepared first by mixing 1% sulfuric acid with 3 drops 5% NaNO2 solution. A 5 drops of the sample was added and 3-5 drops 10% Na2CO3 to the diazo reagent. A red coloration was noted.

neutralized with 1M HCl and this hydrolysate was used for another set of characterization test and

intact protein produced a violet solution which is a positive indication of the Biuret test.

chromatography. Ninhydrin test is for detecting free alpha amino groups. Discussion In Table. 1 it shows the results obtained for the Qualitative Color Reaction of Casein and the Basic Hydrolysis. As seen here amino acids have a variety of chemically reactive groups that can be used to characterize both free amino acid and proteins. The following tests are used to detect presence of amino acid and proteins and distinguish between them. The test for presence of aromatic rings which includes tryptophan and tyrosine is Xanthoproteic test. Although Table No. 1 Results for the Qualitative Color Reaction of Casein and Basic Hydrolysis Color Intact Protein Basic Reactions (Casein) Hydrolysis
Biuret Test Ninhydrin Test Xanthoproteic Test Millon s Test Hopkins-Cole Test Sakaguchi test Nitroprusside Test Fohl s Test Test for Amides Pauly Test Purple solution White precipitate Yellow precipitate White precipitate White precipitate Yellow solution Yellow precipitate Brown precipitate Red to blue litmus paper Red solution Light blue solution Light yellow solution Orange solution Yellow precipitate Light brown solution Light yellow solution Yellow solution Brown solution Yellow solution Orange solution

The only amino acid that is negative for the said test is proline. Its principle is oxidative deamination and decarboxylation. A positive indication of this test would be a blue violet coloration in the solution. Casein and the two other hydrolysates must yield a positive result in this test.

phenylalanine is considered one of them, it will not have a positive result because it is inactive. Nitration of the phenyl group is the principle invoved in this test. There should be a positive visible reaction of yellow to orange solution for this test. Intact proteins, acid hydrolysates and basic hydrolysates are positive for this test.

Millon s test is a test for the presence of tyrosine. Its principle is the complexation reaction between phenolic group and mercury that is found in the Millon s reagent. A positive indication of this test is old rose or red precipitate. Intact proteins and the hydrolysates should be positive in this test.

A. Qualitative Color Reactions

The test for the presence of tryptophan is the HopkinsCole Test. The condensation of indole group with glyoxylic acid and H2SO4 is the principle involved in this test. The formation of purple ring on the surface of the solution is a positive indication of this test. According to the results, it was only the acid hydrolysate is negative for this test because tryptophan cannot be identified

The Biuret test is a general test for proteins and for detecting peptide linkage. The principle involved is complexation reaction. The intact protein should be positive in this test since its peptide linkage is not broken unlike the two other samples that had undergone hydrolysis. As seen in the results, only the

and was destroyed during acid hydrolysis. It becomes black precipitate which is a humin.

intact proteins, acidic and basic hydrolysates have positive results for this test.

Sakaguchi test is a test for the presence of free or intact arginine. Subsequently alkaline or basic hydrolysis destroys arginine and produces ornithine and urea, all the samples are positive but basic hydrolysate should be negative for this test. The reaction of guanido group with napthol and an oxidizing reagent is the primary principle involved for this. A positive indication for this test is a red or orange solution. Only the basic hydrolysate had a light color.

The last test performed was Pauly Test which is the test for Histidine and Tyrosine. A dark yellow to orange solution is its positive outcome.

References 1. Retrieved January 6, 2012, http://www.elmhurst.edu/~chm/vchembook/5 65proteins.html 2. Crisostomo, A., et al. Laboratory Manual in General Biochemistry. C & E Publishing, Inc. Quezon City. 3. Retrieved January 6, 2012, http://www.wisegeek.com/what-is-casein.htm.

Nitroprusside test is used for indicating the presence of cysteine. In this test, cysteine is partially destroyed and it produced a red solution. Its principle is complexation. Intact protein is very positive in this test while the acid and basic hydrolysate is only somewhat positive. The cysteine that is partially destroyed is evident in the results of the experiment.

Fohl s test is a test for sulfur containing proteins. It also indicates the presence of methionine and cysteine because those two amino acids have sulfur in their structures. The principle involved is the fusion followed by ionic interaction. The formation of black precipitate from lead sulfide is the positive outcome for this test. The dark coloration of the samples caused by the Fohl s test indicates that there is sulfur in the intact protein, acid hydrolyste and basic hydrolysate.

The test for amides is used to detect R-groups of asparagine and glutamine. The change in color of litmus paper from red to blue is the positive product for this. Its principle is basic hydrolysis. As seen in the result all