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BMLS 2A (Summer)

Qualitative Test for Proteins (Color Reaction)

Objectives

Objectives

Detect proteins using different tests.

Determine significance of tests to protein hydrolysis.

Determine chemical structures in protein that brings out color change.

Determine positive/negative results.

Know the principles involved in each test.

Tests

Xanthoproteic Test 1 mL egg albumin + 5 drops of conc. HNO3

Heat, then Cool

+ 5 drops NH4OH

Xanthoproteic Test

Principle:
It uses concentrated nitric acid. The test gives a positive result in those proteins with amino acids carrying aromatic groups, especially in the presence of Tyrosine.

Xanthoproteic Test

Present: Proteins with amino acids

carrying aromatic groups,

especially Tyrosine

Xanthoproteic Test

Color of precipitate: Dark yellow (+HNO3)

Orange (+NH4OH)

Hopkins-Cole Test 1 mL egg albumin + 1 mL H-C reagent

Incline + 10 drops pure conc. H2SO4

Hopkins-Cole Test

Principle:
When protein mixed with glyoxylic acid and H2SO4, a violet ring will form beneath the protein mixture.
Due to the presence of an indole nucleus in the tryptophan component. Tryptophan condenses with the aldehyde to form the colored product.

Hopkins-Cole Test

Present: Tryptophan (indole group)

Hopkins-Cole Test

Color: Violet ring between two

liquids

Biuret Test 1 mL egg albumin + 1 mL 10% NaOH

1 mL 0.5% CuSO4, drop by drop

Biuret Test

Principle:
Alkaline solution of proteins treated with copper sulfate solution results in the production of a rose-pink to violet color.

This is due to the presence of a peptide linkage.

Biuret Test

Present: Peptide bonds

(--C=ONH2)

Biuret Test

Color: Purple solution

Ninhydrin Test 1 mL egg albumin + 1 mL 0.1% Ninhydrin(aq)

Heat to boiling

Cool

Ninhydrin Test

Principle:
When protein is boiled with ninhydrin, a blue color is produced.

Due to the presence of the alpha-amino acid.

All proteins are positive except proline.

alpha-amino acid + ninhydrin ---> reduced ninhydrin + alpha-amino acid + H2O alpha-amino acid + H2O ---> alpha-keto acid +NH3 alpha-keto acid + NH3 ---> aldehyde + CO2 alpha-amino acid + 2 ninhydrin ---> CO2 + aldehyde + final complex(BLUE) + 4H2O

Ninhydrin Test

Present: All proteins, peptides,

And amino acids

Except Proline

Ninhydrin Test

Color: Blue solution

Sakaguchi Test 1 mL test solution + 1 mL 10% NaOH

+ 6 drops dilute alcoholic alpha-naphthol solution

Mix
+ 10 drops NaBrO solution

Sakaguchi Test

Present: Arginine (guanidinium group)

Sakaguchi Test

Principle:
It is used for the detection of a specific type of protein with the amino acid containing the guanidinium group to form a red color.

Sakaguchi Test

Color: Red solution

Results

Results
Table 2A 1 Qualitative Test for Ovalbumin Experimental Result Test Test Specificity Theoretical Result Result Drawing Remarks

1. Xanthoproteic Test

Amino acids with a benzene ring

Yellow precipitate

Yellow Precipitate

(+)

2. Hopkins-Cole Test

Tryptophan

Violet ring between the two liquids

Cloudy white Precipitate

(-)

3. Biuret Test

Peptide Bonds

Violet Solution

Violet Solution

(+)

4. Ninhydrin Test

All proteins, peptide bonds and amino acids except Proline

Blue Solution

Blue Solution

(+)

5. Sakaguchi Test

Arginine

Red Solution

Red Solution

(+)

Analysis

Xanthoproteic Test - Analysis

The experiment yielded a positive result when a yellow precipitate formed.
Thus, amino acids with benzene rings (Tyosine, Phenylalanine, Tryptohan) are present.

Hopkins-Cole Test - Analysis

The experiment yielded a negative result when a cloudy precipitate formed instead of a violet ring between the two liquids.
Thus, tryptophan is absent. We accept this as an experimentl error as ovalbumin generally contains a lot of tryptophan.

Biuret Test - Analysis

The experiment yielded a positive result when the solution turned blue-violet.
Thus, many peptide bonds are present.

Ninhydrin Test - Analysis

The experiment yielded a positive result when the solution turned blue.
Thus, many proteins, peptide bonds and amino acids are present. Though it exempts Proline, it is not a guarantee that Proline is not present. Its just that it cannot detect Prolines existence.

Sakaguchi Test - Analysis

The experiment yielded a positive result when the solution turned red.
Thus, the amino acid containing the guanidinium group, Arginine, is present.

Conclusion

Conclusion
Ovalbumin in egg white contains many types of amino acids, be it as residues, connected via peptide bonds, or part of long-chain proteins. That is why eggs are important in the human diet, as it provides the essential amino acids required to run our body in the principle of homeostasis.
There are different tests for different types of amino acids and some tests exclude a specific amino acid. Tryptophan is present in ovalbumin, even if our experimental data showed otherwise. This just means that there are many factors involved in the accuracy of data gathered.