Characterization of Intact Protein and Acid Hydrolyzate from Casein using

Color Reaction Tests

Marrion Khennie I. Que, Leonard Louie E. Pedroso,*Siara Lou B. Sangalang, Chris Allana

Marella B. Tugade

College of Science, University of Santo Tomas, España Blvd., Manila

I. Abstract

Proteins and hydrolysate can be characterized by using color reactions. Proteins

are amino acids linked by peptide bonds. However, in an Acid Hydrolyzate, it
contains free amino acids due to the breaking of peptide bonds. In this experiment,
five different test were utilized. The Biuret test which is used to detect the
presence of peptide bonds. The Ninhydrin test which detects the presence of alpha
amine groups. The Xanthoproteic test which is used to detect the presence of a
benzene ring. The Hopkins- Cole test which is used to detect the presence of
indole ring. Lastly, the Sakaguchi test which is used to detect the presence of
guanido groups.

II. Introduction

Amino acids are the building blocks of proteins and they are attached to each other forming

long chains called proteins. Proteins are complex molecules that are essential to the function,

structure, and regulation of the organs and tissues in our body. They play an important role in the

biological process of living organism. Different proteins are results of different combination of

amino acids and each of them has specific functions.

Milk contains the proteins casein which is approximately 82% and serum which is 18% of

the composition. The protein casein is digested slower than other protein which allows the

prolonged release of amino acids in the blood stream. Due to this, it has different effects to the

human body like weight loss, calorie burning, and it also help in body composition. There are

several types of casein. These types are formed by different sequence of amino acids. The structure

of casein are said to be opened due to high proline content. Casein also has an abundance of

phosphate which allows milk to contain calcium.

 In our body, enzymes are responsible for hydrolyzing protein to improve the function of

protein in our system. Hydrolyzation is the process of breaking down the proteins into amino acids.

In our body, the pancreatic protease is responsible for hydrolysis. Another common method is the

prolonged boiling of protein in strong acids or strong bases.

Proteins can be characterized in different ways one of which is through the color reaction

test. These tests are used to determine the presence of different structures. The different colors are

the result of the peptide bonds present and different compositions of the amino acids. The biuret

test which is also known as Piotrowski Test is used to determine the extent of hydrolysis and the

presence of peptide bonds the positive visible result of this test is a violet solution. The Sakaguchi

Test is used to detect the presence of a guanido group called arginine. A red solution suggests a

positive result for this test. The ninhydrin test is used to detect the presence of an amino group. It

gives a purple color for alpha amino acids and orange color for secondary amines. The

xanthoproteic test is used to determine the presence of benzene rings in the presence of tyrosine,

tryptophan, and phenylalanine . The positive result is a dark yellow solution which is neutralized

with an alkali. Lastly, the Hopkins- Cole test, which is also the glyoxylic acid test which is used

to detect the presence of tryptophan. The concentrated sulfuric acid is used to form the two layers

and the positive result will show a purple interphase between the layers.

III. Methodology

I. Preparation of Intact Protein Suspension

Half of the isolated casein from the previous experiment was used. It was

cut into small pieces and placed into a mortar and was grinded. Drop by drop, ten milliliters

of distilled water was added until fine protein suspension was obtained.
II. Color Reactions

A. Biuret Test

Using a Pasteur pipette, three drops of protein suspension and hydrolysate

were placed in a spot plate separately and one drop of 2.5M NaOH was added to

each. Then, one drop of 0.01M CuSO4 solution was added and mixed. The color of

the solutions was then noted.

B. Sakaguchi Test

Using a Pasteur pipette, five drops protein suspension and hydrolysate was

placed in a spot plate separately. One drop of 10% NaOH and one drop 0.02%

naphthol solution was added to the suspension and hydrolysate. Then, after about

three minutes, one drop of freshly-prepared 2% NaOBr was added. The color of the

solution was then noted.

C. Ninhydrin Test

Using a pipette, 1 milliliters of water was added to ten drops of protein

suspension in a test tube. On a separate test tube, one milliliters of acid hydrolysate

were placed. To each test tube, 0.5 milliliters of 0.1% ninhydrin solution was added.

Both test tubes were heated in a boiling water bath for three minutes then the color

of the solution was noted.

D. Xanthoproteic Test

Using a pipette, 1 milliliters of water was added to ten drops of protein

suspension in a test tube. On a separate test tube, one milliliters of acid hydrolysate
 were placed. Three drops of concentrated HNO3 was added slowly to each test tube

and the color of the solution was noted. Then both test tubes are heated in a boiling

water bath for one minute. Then, the solutions are cooled with flowing water then

concentrated NaOH was added until the solution was alkaline. Red litmus paper

was used to determine if the solution was basic. Then the color of solution is noted.

E. Hopkins-Cole Test

On separate test tubes, two drops of protein suspension and hydrolysate was

placed and one milliliters of Hopkins-Cole reagent to both test tubes. In an inclined

test tube, one milliliters of concentrated H2SO4 was added slowly down the side of

the test tube until two layers form and the color of the interphase was noted.

IV. Results & Discussion

Table 1. Color reactions of Intact protein and acid hydrolyzate

Color Reaction Intact Protein Acid Hydrolysis

Biuret Test Light purple solution Light blue solution

Sakaguchi Test Light red solution with dark
Ninhydrin Test
Xanthoproteic Test
Hopkins-Cole Test
Light orange solution
orange precipitate
Light purple solution with
Cloudy solution
light purple precipitate
1. Turbid solution with
1. Clear solution
yellow precipitate
2. Clear solution
2. Yellow solution
Cloudy interphase White interphase

Discussion:

The biuret test in intact protein showed a light purple solution which is a positive visible result for

the test. The Copper(II) which is present in the reagent reacts with the nitrogen in the peptide bond

which gave the violet solution however it will give a colorless or a blue solution if the there is no
peptide bond present. In the experiment, the intact protein resulted in a purple solution while the

hydrolyzate had a blue solution.

In the Sakaguchi test, a red solution with dark orange precipitate was observed for the intact protein

while a light orange solution was observed in the acid hydrolysate. The napthanol and the oxidizing

agent, sodium hypobromine, in the reagent reacts with arginine present in the protein which gave

a red solution as a visible positive result. It was observed that there is a red solution in the intact

protein and a light orange solution in the hydrolysate which suggest the presence of arginine in

both. Although the desired outcome was reached for the intact protein, the hydrolyzate contains

arginine as suggested in the change of color of the solution from clear to light orange. There might

have been an error in the process of hydrolyzation which had caused the reaction.

In Ninhydrin test, a light purple solution with light purple precipitate was observed in the intact

protein while a cloudy solution was observed in the hydrolysate. The reaction between the amino

group and the ninhydrine gave a purple solution. This is true to all amino groups except for proline

and hydroproline which will give a yellow solution. In the experiment, purple solution with a

purple precipitate was observed which is a positive result while in the hydrolysate, a cloudy

solution was observed which means no reaction took place.

The Xanthoproteic test give a positive result of a yellow solution due to the nitration of the

aromatic benzene. A positive result on this test suggests the presence of an aromatic nucleus. Upon

adding alkali, the yellow solution turns from yellow to orange. In the experiment, a turbid solution

which turns into yellow solution once the alkali was added was observed in the intact protein which

suggested a positive result while the hydrolysate maintained a clear solution which means no

reaction took place.

The Hopkins-Cole test aims to detect the presence of tryptophan which is an indole group. This

indole group reacts with glyoxylic acid. Upon adding the sulfuric acid, two layers shall be formed

with a purple ring in the interphase. In the experiment, both the intact protein and the hydrolysate

gave cloudy or white interphase which suggest a negative reaction. This is not the desired result

for the intact protein. There may be an error in the preparation of the intact protein which resulted

for the observation.

V. Conclusion

In conclusion, the intact protein suspension gave a positive result for the Biuret, Sakaguchi,

Ninhydrin, and Xanthoproteic tests. The result suggest the presence of peptide bonds, argine,

alpha amino acid, and an aromatic ring in the intact protein. The acid hydrolysate gave a positive

result in Sakaguchi test only which confirms the presence of arginine.

 VI. References

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