QUALITATIVE COLOR REACTIONS OF INTACT CASEIN AND ALBUMIN AND THEIR ALKALINE HYDROLYSATES

Berberabe, Janel Elane R.; Bungato, William D.; Caluag, Janssen C.; Cambri, Ma. Victoria Claudine M.; Cerado, Alexis U. Group 2 2A Pharmacy General Biochemistry Laboratory

ABSTRACT
Violet coloration in Biuret test denoted the presence of peptide bonds; blue/blue-violet coloration in Ninhydrin test denoted the presence of an -amino acid, while the change in color in the Xanthoproteic test simply denoted the presence of side chains for aromatic amino acids. A change in color in Millons test denoted the presence of residues of the amino acid tyrosine, while the formation of a violet ring in Hopkins-Cole test denoted the presence of residues of tryptophan. A red coloration in Sakaguchi test denoted the presence of arginine (guanido group), while the formation of a red solution for Nitroprusside test denoted the presence of cysteine. A change in color in the red litmus paper (red to blue) in the test for amides simply denoted the presence Rgroups of aspargine and glutamine. The formation of a dark sediment in Fohls test denoted the presence of sulfurcontaining amino acids while Pauly test confirms for the presence of tyrosine and histidine residues when a red coloration or purple coloration is observed.

INTRODUCTION
The intact protein samples (casein and albumin) extracted from skimmed milk were tested for the presence of the different amino acids and each subsequent result was recorded. In separate test tubes, 0.5 grams of intact casein (dissolved in 1 mL of distilled water) and 5 drops of intact albumin (with 1 mL distilled water) were prepared. The same was done for their respective alkaline hydrolysates, with the test tubes containing 0.5 mL of the hydrolyzed sample. These test tubes were then subjected to each different tests to be able to determine some of their amino acid compositions.

EXPERIMENTAL
A. Compounds tested Intact casein and albumin, hydrolysates of casein and albumin alkaline

B. Procedure 1. Biuret Test 20 drops of 2.5 M NaOH were added to each of the sample preparations (mixed well). Afterwards, 2-3 drops of 0.1 M CuSO4 were added. The test tubes were then shaken to properly observe the color result. 2. Ninhydrin Test 6-10 drops of 0.1% Ninhydrin solution were added to the sample preparations, then the test tubes were placed in a boiling water bath. 3. Xanthoproteic Test 10 drops of conc. HNO3 were added to the samples (mixed well), resulting to the first coloration. Afterwards, 10 drops of conc. NaOH were added to the preparation, resulting to the second coloration. 4. Millons Test 5 drops of Millons reagent were added to the sample preparations and the change in color was noted.

5. Hopkins-Cole Test 20 drops of Hopkins-Cole reagent were added to the sample preparations. The test tubes were then inclined and 20 drops of conc. H2SO4 were added slowly along the sides of the test tubes. Without shaking the mixture, the color change was noted. 6. Sakaguchi Test 10 drops of 10% NaOH and 10 drops of 0.02% Naphthol solution were added to the sample preparations, mixed, and let stand for a minute. 3 drops of 2% NaOBr were then added to the preparations, mixed, and the color produced was noted. 7. Nitroprusside Test 0.5 mL of 3 M NaOH was added to 0.5 mL of each of the sample preparations. 0.25 mL of 2% Nitroprusside solution was then added to the preparation and a formation of red solution was noted. 8. Fohls Test 5 drops of 30% NaOH and 2 drops of 5% (CH3COO)2Pb were added to each of the sample preparations. The test tubes were then placed in a boiling water bath, and a formation of a dark sediment was noted. 9. Test for Amides 2 mL of 20% NaOH to 10 drops of each of the sample preparations, then the test tubes were placed in a boiling water bath. Evolution of gas was tested by placing a moistened red litmus paper over the mouth of the test tubes. 10. Pauly Test A diazo reagent was first prepared by mixing 3-5 drops of 1% sulfanilic acid with 3 drops of 5% NaNO2 solution. 5 drops of the sample preparation with 3-5 drops of 10% Na2CO3 were added to the diazo reagent previously prepared, and the appearance of a red coloration was noted.

RESULTS AND DISCUSSION

1. Biuret Test The Biuret test is used to determine the presence of peptide bonds in each of the protein samples. A violet/blue-violet coloration is considered as the positive result for the said test. Table 1. Results for the Biuret Test
Intact proteins Casein Albumin (+) (+) Violet Violet coloration coloration Alkaline hydrolysates Casein Albumin (-) (-) Colorless/no Blue change coloration

Table 3. Results for the Xanthoproteic Test

Intact proteins Casein Albumin Dark yellow Light yellow coloration coloration Alkaline hydrolysates Casein Albumin Dark Dark orange orange coloration coloration

The violet coloration observed in the intact proteins denoted the presence of peptide bonds, while the lack of color change in the alkaline hydrolysate of casein and the blue coloration in the alkaline hydrolysate of albumin denoted the absence of peptide bonds in the samples, since the process of hydrolyzing the proteins broke the peptide bonds initially present in the sample. 2. Ninhydrin Test The Ninhydrin test is used to determine the presence of an -amino acid in each of the protein samples, hence, it will yield positive results for protein samples containing lysine and/or arginine in their protein structures. A blue/blue-violet coloration is considered as a positive result for the said test. Table 2. Results for the Ninhydrin Test
Intact proteins Casein Albumin (+) (+) Blue-violet Blue-violet coloration coloration Alkaline hydrolysates Casein Albumin (+) (+) Violet Violet coloration coloration

Changes in color for the Xanthoproteic test confirmed the presence of side chains in aromatic amino acids. Based on the results, the alkaline hydrolysates of the casein and albumin both yielded colors of higher intensity, which can be interpreted as the hydrolysates simply having more aromatic rings compared to the intact proteins. 4. Millons Test Millons test is used to detect the presence of a phenolic group/phenolic compounds present in an amino acid, and a pinkish/rose coloration is considered as the positive result for this test. A sample containing tyrosine will produce a positive result for the said test. Table 4. Results for Millons Test
Intact proteins Casein Albumin (+) (+) Pinkish Pinkish coloration coloration Alkaline hydrolysates Casein Albumin (-) (-) Yellow Yellow solution solution

The colorations observed for Ninhydrin test denoted the presence of an -amino acid in each of the protein samples and their hydrolysates, the only difference being the intensity of the color produced. The alkaline hydrolysates produced a darker shade of violet hue compared to their respective intact proteins. This could be due to the presence of ammonia or primary or secondary amines on the hydrolysates. 3. Xanthoproteic Test The Xanthoproteic test is used to detect the side chains of aromatic amino acids. It yields positive results with substances possessing tryptophan and tyrosine in their respective structures. A change in color (yellow, orange) is considered as the positive result for this test.

The intact proteins showed the pinkish red coloration expected for a positive result, hence the presence of tyrosine in each of them. Their alkaline hydrolysates, meanwhile, yielded negative results for the said test, denoting the absence of tyrosine. 5. Hopkins-Cole Test The Hopkins-Cole test is used to determine the presence of the amino acid tryptophan. The positive result for the said test is the formation of a violet ring or simply a violet coloration. Table 5. Results for the Hopkins-Cole Test
Intact proteins Casein Albumin (+) (-) Formation Colorless of a violet solution/no ring change Alkaline hydrolysates Casein Albumin (+) (-) Formation Colorless of a violet solution/no ring change

The formation of a violet ring in casein denoted the presence on an indole ring, which is contained only in the amino acid tryptophan. Only casein and its alkaline hydrolysate formed the expected positive result, thus confirming the presence of tryptophan. Albumin and its alkaline

hydrolysate had no color change, hence the absence of tryptophan. 6. Sakaguchi Test Sakaguchi test is used to determine the presence of arginine in a protein sample, since arginine is the only amino acid containing a guanido group in its structure. specifically, the test yields a positive result of orange-red/wine red coloration for the presence of a guanido/guanidino group in the sample. Table 6. Results for Sakaguchi Test
Intact proteins Casein Albumin (+) (+) Red Red coloration coloration Alkaline hydrolysates Casein Albumin (-) (-) Colorless/no Colorless/no change change

Table 8. Results for Fohls Test

Intact proteins Casein Albumin (+) (+) Formation Formation of a dark of a dark brown brown sediment sediment Alkaline hydrolysates Casein Albumin (-) (-) Formation Formation of a dark of a dark brown brown sediment sediment

The intact proteins underwent a change in coloration when the Sakaguchi test was performed on them, hence, the presence of arginine in all the sample substances. Their alkaline hydrolysates, however, yielded negative results, since arginine was hydrolyzed to ornithine and urea, hence, absent in the hydrolyzed samples. 7. Nitroprusside Test The Nitroprusside test is used to determine the presence of cysteine. The positive result for this test is the formation of a red solution. Table 7. Results for the Nitroprusside test
Intact proteins Casein Albumin (+) (+) Red Red solution solution Alkaline hydrolysates Casein Albumin (-) (-) Yellow Yellow solution solution

All the sample proteins and their respective hydrolysates yielded positive results for the said test, hence confirming the presence of sulfur-containing amino acids in each sample. 9. Test for Amides The test for amides is used to detect the R-groups of aspargine and glutamine. It also indicates the presence of primary, secondary and tertiary amides and nitriles. A change of color of the red litmus paper to blue is considered as the positive result for this test. Table 9. Results for the test for amides
Intact proteins Casein Albumin (+) (+) Litmus Litmus paper paper changed changed from red to from red to blue blue Alkaline hydrolysates Casein Albumin (+) (+) Litmus Litmus paper paper changed changed from red to from red to blue blue

The Nitroprusside test yielded positive results only for the intact proteins, thus confirming that cysteine present was destroyed during alkaline hydrolysis of both casein and albumin. 8. Fohls Test Fohls test (also called the Lead Acetate test or reduced sulfur test) is used to determine the presence of sulfur-containing amino acids, hence the presence of cysteine in any of the protein structures. Methionine, on the other hand, has a sulfur present in its structure, but it is not readily removed by alkali, hence, it is not affected by the chemical reaction taking place in the test. The formation of a dark sediment/precipitate (black or brown) is considered as the positive result for this test.

All sample substances, upon exposure to the moistened red litmus paper changed their color from red to blue, hence confirming the presence of amides. 10. Pauly Test The principle involved in Pauly test is diazotization, in which the sulfanilic acid undergoes the diazotization process (due to the addition of NaNO3 and Na2CO3). The red coloration after addition of NaNO3 and Na2CO3 confirms the presence of tyrosine and histidine residues. Table 10. Results for Pauly Test
Intact proteins Casein Albumin (+) (+) Red Red coloration coloration Alkaline hydrolysates Casein Albumin (+) (+) Red Red coloration coloration

The intact proteins as well as their alkaline hydrolysates yielded positive results for the said test, thus confirming the presence of tyrosine, and histidine residues in their respective structures.

REFERENCES
From books Shankara, S. (2008). Laboratory Manual for Practical Biochemistry. New Delhi: Jaypee Brothers Medical Publishers. Litwack, G. (1960). Experimental Biochemistry: A Laboratory Manual. New York: Wiley From the internet Purchon, N. Virtual Laboratory: Protein Test. http://www.purchon.com/biology/protein.htm 1/15/12 Ophardt, C. Virtual Chem Book: Proteins Introduction. http://www.elmhurst.edu/~chm/vchembook/565 proteins.html 1/15/12 Wang, N. Digestion of protein into an amino acid. http://www.eng.umd.edu/~nsw/ench485/lab3.ht m 1/15/12 From scientific journals Lawrence, K., Creamer, K. L., et al., General Properties of Casein. Journal of Dairy Science 81 (1998) 3004-3012.