EXPERIMENT 3
PROTEIN AND AMINO ACIDS TEST
1.0 OBJECTIVE
To determine qualitatively and quantitatively presence or absence of protein and amino
acid in a sample
2.0 CORRESPONDING COURSE OUTCOME
Ability to describe and differentiate the role of proteins and enzymes in biochemistry and
purification of proteins (CO3)
3.0 INTRODUCTION
There are various qualitative tests for detection of amino acids in a samples preparation.
Some of these tests are general and are given by all the amino acids while others are
specific for a particular amino acid. The latter type of tests can thus be employed for
identification of particular amino acid. Many of these tests give positive response
irrespective of the fact whether the concern amino acid exists in free form in a solution or
happens to be constituent of protein molecule. Hence, these tests can also be employed
for the purpose of detection of protein in a sample. A negative test for a particular amino
acid, however, need not necessarily denote absence of protein in a purified sample
preparation. Such a situation can be encountered if the isolation protein totally lacks or
contains very few residue of that particular amino acid. For example, failure to get a
positive result for Hopkins-Cole test (which is specific for tryptophan) for purified
preparation of gelatin is not due to absence of protein but because this protein does not
contain any tryptophan residue. However, all proteins do give positive reaction with
ninhydrin and biuret and thus serve as reliable qualitative test for preliminary
establishment of present of protein in a sample.
3.1 Biuret Test:
This test is a general test for compounds having a peptide bond. Alkaline copper sulfate
reacts with compounds containing two or more peptide bonds to give a violet or pinkish
coloured product which due to formation of coordination complex of cupric ions with
unshared electron pairs of peptide nitrogen and oxygen of water. The illustrated reaction of
Biuret reagent with compounds contains two or more peptide bond are shown in Figure 2.
Figure 2: Reaction between Biuret reagents with compounds contains two or more peptide
bond
3.2 Xanthoproteic Test:
Amino acids containing an aromatic nucleus (tyrosine, tryptophan, phenylalanine) form
yellow nitro derivatives on heating with concentrated HNO3. The salts of these derivatives
are orange in colour. Protein containing these amino acids also gives a positive response
to this test.
Figure 3: Reaction between xanthproteic reagent with amino acid contain an aromatic
nucleus.
Other colour tests for various amino acids are listed in Table 1.
Table 1: Colour test for proteins and various amino acids
Test/reaction Reactants Specificity of the test Colour obtained
Biuret Alkaline CuSO4 Peptides, protein Purple
Xanthoproteic Boiling conc HNO3 Tyrosine Yellow in acids which turn
reaction Tryptophan orange yellow
Phenylalanine (give
weak test)
4.0 MATERIALS AND EQUIPMENTS
4.1 40% NaOH
4.2 Biuret reagent
4.3 Concentrate HNO3
4.4 Casein
4.5 Gelatin
4.6 Tryptophan
4.7 Tyrosine
4.8 Test tube
4.9 Test tube rack
4.10 Waterbath
5.0 PROCEDURES
5.1 QUALITATIVE TESTS FOR AMINO ACIDS AND PROTEIN
5.1.1 For each sets of tests you will give 0.5% casein, 0.5% gelatin, 0.5% tryptophan
and 0.5% tyrosine test solution. Prepare 1 ml of each solution in different test tube
and test accordingly as follows.
5.1.2 Biuret Test:
To each of the test tubes, add 5 drops copper sulphate solution followed by
1 ml of 40% NaOH.
Mix thoroughly.
The development of a purplish violet colour is evidence of the presence
of proteins.
5.1.3 Xanthoproteic Test:
To each of the test tubes, add 10 drops concentrated HNO 3 while swirling.
Cool and observe the colour change.
Add sufficient 40% NaOH to make the solution strongly alkaline.
A yellow colour in acid solution which turns bright orange with alkali
constitutes a positive result.
(NOTE: Be careful when handling concentrated acid. Do this experiment in fume
hood and wear goggle).
6.0 RESULT AND DISCUSSION
6.1 QUALITATIVE TESTS FOR AMINO ACIDS AND PROTEIN
Table 3: Colour reaction from various tests for proteins and amino acids
Biuret Test Xanthoproteic Test
0.5% casein
0.5% gelatin
0.5% tryptophan
0.5% tyrosine
7.0 REFERENCE
Sawney, S.K and Randhir, R. (2005). Introductory Practical Biochemistry. Alpha Science,
Harrow, UK.
BIOMEDICAL ELECTRONICS ENGINEERING PROGRAMME
SCHOOL OF MECHATRONIC ENGINEERING
ENT 210 BIOCHEMISTRY
EXPERIMENT 3: PROTEIN AND AMINO ACIDS TEST
Name :
Matric No. :
Group Members :
Date of experiment:
Date of submission:
INTRODUCTION:
THEORY:
RESULT:
Table 1: Colour reaction from various tests for proteins and amino acids
Biuret Test Xanthoproteic Test
0.5% casein
0.5% gelatin
0.5% tryptophan
0.5% tyrosine
DISCUSSION & CONCLUSION:
