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 HNO 3. 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 CuSO 4 Peptides, protein Purple Xanthoproteic Boiling conc HNO 3 Tyrosine Yellow in acids which turn reaction Tryptophan Phenylalanine (give weak test) orange yellow 4.0 MATERIALS AND EQUIPMENTS 4.1 40% NaOH 4.2 Biuret reagent 4.3 Concentrate HNO 3 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: