1.

6 QUALITATIVE COLOUR REACTION OF INTACT AND HYDROLYZED PROTEIN PRODUCTS

Proteins give various colour reactions which are not specific for protein molecules, but for
certain groups of the component of amino acids. A number of qualitative colour reaction have been
devised which are useful for the detection of proteins. Qwing to the complexity of the protein molecule
and the difficulty of obtaining a single pure protein compound, these tests are to be used with
knowledge that they test for specific chemicals groupings on the protein structure, or more generally for
the groupings which most proteins contain.
Objective: To qualitatively identify the presence of amino acids of the different protein sources in the
different test
Materials:
a. 10% NaOH solution REAGENTS:
a. 1% Ninhydrin solution
b. Concentrated NaOH solution
c. 1% CuSO4 solution b. Millon’s reagent
d. 1% casein solution c. Hopkin’s cole reagent
e. 1% Gluten d. 0.02% naphthol solution
f. 1% bean protein
g. 1% egg albumin
h. 1% myoglobin
i. Concentrated nitric acid
j. Concentrated sulphuric acid
k. 2% NaOBr/ NaOCl
l. 1% lead acetate solution
Note: (1g casein in 100mL of either 1.5 M Tris Buffer (pH 8.8) or PBS 1x (pH 7.4) dissolved at 65oC)
GENERAL TEST:
1.7.1 BIURET TEST
1. Add 1 mL of 10% NaOH to 3mL of the protein suspension. Mix.
2. Add a drop of 1% CuSO4 solution.
3. Mix if no definite color develops, add another drop or two of the 1% CuSO4 solution.
4. Take note the intensity of the color change.
PRINCIPLE:
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Reference/s:
1.7.2 NINHYDRIN TEST
1. Add 0.5mL of 1% Ninhydrin solution to 3mL of the protein solution.
2. Heat to boiling for 1 to 2 minutes.
3. Allow to cool.
PRINCIPLE:
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Reference/s:
SPECIFIC TEST
1.7.3 XANTHOPROTEIC TEST
1. Add slowly 1mL concentrated HNO3 to 3mL of the protein suspension.
2. Heat gently to boiling for 30 seconds. Then, cool the mixture with flowing water.
3. Add slowly drop by drop concentrated NaOH until excess or the solution is lightly alkaline.
4. Note the intensification of color.

PRINCIPLE:
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Reference/s:

1.7.4 MILLON’S TEST

1. Add 5 drops of fresh Millon’s reagent to 3mL suspension.
2. Carefully heat the mixture to boiling for 30 seconds.
3. If too much reagent has been used, the color may disappear on boiling.
4. Note any changes that occur.
PRINCIPLE:
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Reference/s:
1.7.5 HOPKINS- COLE TEST
1. Add 2mL of Hopkins-cole reagent to 3mL of protein suspension. Mix.
2. Incline the tube and add slowly 5mL of concentrated H2SO4 down the side of the tube so that
the
layers form.
3. Note the color formed at the junction of the two layers.
4. It might be necessary to rotate the tube gently to develop the color at the interphase.
PRINCIPLE:
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Reference/s:
1.7.6 SAKAGUCHI TEST
1. Add 1mL of 10% NaOH and 3mL of the 0.02% naphthol solution to 5mL cold protein
suspension.
2. After 3 minutes add 2-4 drops of freshly prepared 2% NaOBr/ NaOCl. Note the color.
3. Stabilize the color by adding urea to destroy the excess hypobromite/ hypochlorite.
PRINCIPLE:
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Reference/s:

1.7.7 LEAD ACETATE TEST

1. Add 1mL of 10% NaOH to 3mL of protein suspension.
2. Heat to boiling water bath for 5-10 minutes. Cool.
3. Add few drops of 1% lead acetate solution.
PRINCIPLE:
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Reference/s:
Name: _________________________________ Group No:
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Schedule: ______________________________ Date:
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ACTIVITY NO. 1.7.1-1.7.7

QUALITATIVE COLOR REACTION OF INTACT PROTEINS – Answers to post laboratory questions
1.7.1 BIURET
1. Explain the intensity of the color change. What is the reactive group responsible for the
positive biuret test?
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Reference/s:

1.7.2 NINHYDRIN
1. What amino acids won’t give a typical reaction in the ninhydrin test? Why?
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Reference/s:

1.6.3 XANTHOPROTEIC TEST

1. What 3 amino acids will produce positive xanthoproteic test and what functional group of
the amino acids is responsible?
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Reference/s:

functional group in these amino

TESTS POSITIVE AMINO ACIDS PRESENT acids is responsible for the
positive reaction
Millon’s test

Hopkins- cole test

Sakaguchi test

Lead acetate test