Practical 5: Detection of protein and protein denaturation

Proteins are intrinsic components of most foods and are also added in purified form during
processing to perform certain desirable functions. From the perspective of the food scientist,
functional properties of proteins are those properties that influence food quality and appeal.
These properties are determined by the primary, secondary, tertiary and quaternary structures of
proteins and vary widely among proteins. Heating, changes in pH, whipping, drying and other
treatments may alter the functional properties of proteins.
Apparatus and Equipments
Test tubes
Test tube rack
Glass dropper
Reagents and Materials
CuSO4, 1%
NaOH, 2 M
HCl, 2 M
Protein (albumin or gelatin) solution, 1%
NaOH, 0.2 M
HCl, 0.2 M
Distilled water
Concentrated HNO3
Concentrated H2SO4
Concentrated HCl
NaOH, 40%
Red methyl indicator (pka 5.5)

(A) Quantitative test for Protein-Biuret test

This is general test for peptide bond, all proteins in general respond positively to the Biuret test.
Proteins containing two or more peptide bonds react with alkaline copper sulphate to produce a
purple colored product, which is developed due to the formation of a cupric-coordination
complex.
1. Transfer 2 ml of the test sample into a test tube.
2. Add 5 drops of copper sulphate and 2 ml of 2 M NaOH.
3. Mix the contents and observe the tube for the development of a purple color, which
indicates the presence of peptide bonds, hence that of proteins.

(B) Effect of Heat

1. Prepare and label three test tubes (1, 2 and 3).
2. Add 2 ml of albumin solution and at 2 drops of red methyl indicator.
3. For test tube 1, add 2 ml distilled water.
4. For test tube 2, add 2 ml 0.2 M NaOH.
5. For test tube 3, add 2 ml 0.2 M HCl.
6. Color for test tube 1 should be orange; if yellowish, add extra HCl drop by drop until
orange color. For test tube 2 and 3, adjust the pH by adding HCl or NaOH, until obtain
the same color as test tube 1.
7. Place the test tubes in boiling water bath for 5 minutes.
8. Observe the change and record it.
9. Repeat Steps 1-8 for gelatin solution.

(C) Effect of pH
1. Transfer 2 ml of albumin into test tube.
2. Slowly add in concentrated HNO3 (through the wall of test tube) to form a layer under the
albumin solution. This is also known as Heller's Ring and it is used to clinically detect
the presence of albumin in urine.
3. Shake the test tube carefully to mix the mixture. Observe and record the change.
4. Repeat Steps 1-3 with concentrated H2SO4, concentrated HCl and 40% NaOH.
5. For gelatin solution, transfer 2 ml of gelatin into test tube and repeat Steps 1-4.

References
1. Miller, D. D. (1998). Food chemistry: A laboratory manual. New York: WileyInterscience.
2. Nigam (2007) Lab Manual in Biochemistry: Immunology and Biotechnology: Tata
McGraw-Hill Education.