ISOLATION AND CHARACTERIZATION OF PROTEINS

(Alkaline Hydrolysis of Gluten)

Group#5 2APH Mia Sheleth V. Manalo, Kimberly I. Manlangit, Jefferson B. Manuel, Shirmagne Fatima E. Manugas, Jessieca I. Ongsitco

ABSTRACT

Gluten can be defined as the rubbery mass that remains when wheat dough is washed to remove starch granules and water-soluble constituents.Gluten was extracted and hydrolyzed by base and subjected to different qualitative tests. Paper chromatography had been done to determine and analyze the different amino acid components of the protein. Results were all positive indicating that gluten contains many amino acids in it.

INTRODUCTION

Proteins can be considered as polymers of

amino acids. Amino acids are linked by

covalent peptide bond into linear chain,

which is called peptide or polypeptide chain.

The properties common to all amino acids

are due to the relative special arrangements

of the carboxyl and amino groups. The

physical and chemical properties unique to

each amino acid are the result of the

structure and chemical properties of the R

group. Several amino acids combine to form

peptide bonds. Proteins contain polypeptide

units (several peptide units). When a protein

is hydrolyzed, it breaks down into smaller

units (tri and dipeptides), eventually forming

amino acids. Specific reactions are used for

the purpose of identifying amino acids and

proteins in biological media, for qualitative

and quantitative analysis. The biuret test is

used to detect the presence of peptide bonds

while the Ninhydrin reaction is a typical test

for an α-amino acid. Xanthoproteic test

detects side chains of aromatic amino acids

while the Millon’s and Hopkins-Cole tests

determine tyrosine and tryptophan residues,

respectively. The Nitroprusside test is used

to find out if sulfur-containing amino acids

are present; test for amides is used to detect

R-groups of asparagine and glutamine. Test

for amides is used to detects R-groups of

asparagines and glutamine.

The objective of the experiment is to determine the amino acid components of gluten which can be done by partition paper chromatography which is widely employed for the separation of amino acids. The solvent migrates along a strip of paper and carries amino acid dissolved in it.

METHODOLOGY

Alkaline Hydrolysis of Intact Protein

Gluten extracted from wheat flour was hydrolyzed by base after it was tested with different qualitative color tests. The alkaline hydrolysis of protein undergone by adding 10 mL of 4 M NaOH to 0.5 g isolated protein in a hard glass test tube and then autoclaved for 5 hours (15 psi). After the appearance of the mixture was noted, ten

mL of distilled water was added and was transferred into a 250-mL beaker. The mixture was neutralized with 1 M HCl, and was tested with red and blue litmus paper to check if it was already neutral. And the neutralized mixture was used as a sample for characterization tests and chromatography.

Qualitative Color Reactions

The gluten hydrolysate was tested with different characterization reagents available in the laboratory namely: Biuret, Ninhydrin, Xanthoproteic, Millon’s Hopkins – Cole tests, and Test for Amides. Six test tubes were prepared for each of the test reactions. Each test tube consists of 1 mL of distilled water added to 0.5 mL of hydrolyzed samples.

In Biuret test, 2-3 drops of 0.1 M CuSO4

solution was added to a mixture of the sample and 20 drops of 2.5 M NaOH. The test tube was shaken and the color changes in the solution were observed. For Ninhydrin test, 6-10 drops of 0.1% ninhydrin solution was placed into the sample and then heated in a boiling water bath. The appearance of violet coloration was taken note of. In the Xanthoproteic test, 10 drops of concentrated nitric acid and concentrated sodium hydroxide was slowly added to the diluted sample and then mixed. Color changes after each addition was observed. For the Millon’s test, 5 drops of Millon’s reagent was added to the diluted sample, and the color changes were noted. For the Hopkins-Cole test, 20 drops of Hopkins-Cole reagent was slowly added to the sample, and then mixed. Concentrated sulfuric acid of about 20 drops was slowly added along the side of the test tube, shaken, and the color of the interference was taken note of. Lastly, test for amides was done by adding 1 mL of 20% NaOH to 10 drops of the sample and placing the tube in a boiling water bath. Moistened red litmus paper was placed over the mouth of the tube to test the evolution of gas during heating. Results were taken note of.

Separation and Identification of Amino Acid by Paper Chromatography

A 1.5 cm margin from one edge of the paper was measure and drawn lightly with a pencil on the prepared 12 x 15 cm Whatman filter paper # 1. Five equidistant points were labeled on the line for application of the chosen samples. The samples were air-dried between applications by a capillary tube. The paper was then rolled into a cylinder without overlapping, and then stapled. It was then placed in a pre-equilibrated chamber, and covered to allow the solvent to ascend undisturbed. When the solvent reach at least 2 cm from the other end, the paper was removed and the solvent front was marked immediately with a pencil line. The paper was air-dried and sprayed lightly with 1% ninhydrin reagent. Then, it was oven-dried and the presences of blue, purple, or yellow spots were noted. The spots were encircled with a pencil and the Rf values were computed.

RESULTS AND DISCUSSIONS

Qualitative Color Tests for Hydrolyzed Gluten

Table shows the comparison of the results of color test reactions done in the hydrolyzed protein to that of tests done in intact protein.

Table 1. Results of Qualitative Color Reaction of Intact and Hydrolyzed Gluten.

Color Reaction

Intact Hydrolyzed

Biuret Violet-blue in color

Green ppt

Ninhydrin Blue-violet in color

Violet soln

Xanthoproteic

Orange in color

Yellow clear soln

Millon’s Flesh/peach in color

Flesh/peach soln

Hopkins-Cole Violet-black ring

Violet ring

Test for Amides

Red Blue

Red Blue

Paper Chromatography Analysis of Gluten

Characterization of gluten using paper chromatography yielded different results compared with color tests of the gluten hydrolysate. Table 2 shows the Rf values of standards and the hydrolysate.

Table 2. Rf values of amino acid standards and hydrolyzed gluten

Amino Acid Standards

Rf Values of the SpotsStandards Hydrolysate

Arginine 0.51 0.3429Histidine 0.60 0.3571Glycine 0.59 0.3714Alanine 0.67 0.3857Proline 0.73 0.4

DISCUSSIONS

Subjecting gluten in alkaline hydrolysis which is composed of extreme temperature caused the breaking down of peptide bonds.

Biuret test is used to look the presence of peptide bonds. Because biuret solution contains Cu ions which react with peptide bonds, intact protein will yield a positive result (violet solution) and a completely hydrolyzed protein will display a negative result (green ppt).

Ninhydrin reacts with ammonia, a primary amine, or a secondary amine (amino acids have a primary or alpha amino group, except for proline which has a secondary amino group). They all turn purple/blue right away upon heating with ninhydrin. 

Some amino acids contain aromatic groups that are derivatives of benzene. These aromatic groups can undergo reactions such as the nitration of benzene ring with nitric acid. This nitration, when used to identify the presence of an activated benzene ring, is commonly known as the Xanthoproteic test, because the product is yellow. The intensity of the yellow color deepens when the reaction occurs in basic solution. Intact protein resulted positive and since histidine and proline are amino acids, hydrolyzed protein yielded a positive result also.

Millon’s test is a test specific for tyrosine, the only amino acid containing phenol group. In this test, the phenol group of tyrosine is first nitrated by nitric acid in the test solution. Then the nitrated tyrosine complexes Mercury (I) and Mercury (II) ions in the solution to form red precipitate or red solution. And since gluten does not contain tyrosine, both intact and hydrolyzed protein when tested resulted negative in the color reactions and chromatography.

The Hopkins-Cole test is specific for trypthopan, the only amino acid containing an indole group. And gluten when tested resulted into an indole violet ring which indicates the presence of trypthopan.

Test for amides uses litmus paper to show whether the protein contains amides.

Chromatography is a technique that separates mixtures into their individual components for example: if we put black washable ink onto a tissue, the ink will spread outwards from the place where we blotted it however, the various components of the ink can't all move at the same speed as it spreads out - so the components will visibly separate.

As we can see from the results, Arginine, Histidine, Glycine, Alanine and Proline are present.

The values in the table has its discrepancy due to errors done while doing the experiment.

The thing that is measured in chromatography is the difference between how far a substance (from the mixture) travels compared to how far the solvent travels.

· Rf = (distance traveled by a substance) / (distance traveled by the solvent)

The experiment then, successfully characterized the different amino acid components of gluten by using the qualitative color reaction tests and paper chromatography.

REFERENCES

The Biochemistry Department (2010). Laboratory Manual in General Biochemistry. Manila: University of Santo Tomas.

“Gluten” En.wikipedia.org/wiki/Gluten

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