Ready Review Characteristics of Life, Cell Structure & Organization by Brilliant Biology Student
Ready Review Characteristics of Life, Cell Structure & Organization. The first in a series of review books for Human and Social Biology. The content is concise, yet comprehensive, so that you can use your study time efficiently.
Biuret Test for Protein
The Biuret Test is often used to determine the presence of peptide bonds in protein. At this level you will be testing for the presence of protein in foods.
The test may also be extended to quantitatively measure the concentration of total protein.
Instead of the Biuret Reagent, either of the following may be used:
1. Add 2 cm3 of the liquid food sample* to a clean, dry test tube2. Add 2 cm3 of Biuret Reagent.
ALTERNATIVELY: Use sodium hydroxide solution and copper sulphate solution instead. Add 1 cm3 of sodium hydroxide solution (40% or bench solution) and 1% copper (II) sulphate solution dropwise – drop by drop - to the food sample
OR Use Fehling's A and B solutions instead. Fehling's A and B should be freshly prepared. Fehling’s A is copper (II) solution and Fehling's B is a mixture of sodium potassium tartrate and sodium hydroxide solution. 1cm3 of each solution – A and B – should be added to the food sample.
3. Repeat steps 1 and 2 with de-ionized water to prepare a negative control and with albumin (egg white) to prepare a positive control.
4. Shake well and allow the mixture to stand for 5 minutes.
5. Observe any color change.
* Prepare liquid samples from solid foods. Crush the solid food, add a little de-ionized water and decant the liquid. This liquid should be used for the food test. The quantity of food crushed and water used depends on the number of tests to be conducted.
Observations and Interpretation
Principle of the Biuret Test
The Biuret test is based on the ability of Cu (II) ions to form a violet-coloured chelate complex with peptide bonds (-CONH-groups) in alkaline conditions.
BIURET REAGENT *
Biuret Reagent contains:
*The biuret reagent is not named after someone but a substance called biuret (H2NC(O)NHC(O)NH) – the result of the condensation of 2 molecules of urea. The reagent is so named because the peptide bonds in biuret give a positive result for the test.
The peptide bond is found between the carboxyl and amino groups of 2 adjacent amino acid residues.
Peptide bonds are found in peptides, polypeptides and proteins, all of which give a positive result for the biuret test and are collectively known as protein.
A peptide or a peptone is a short chain of amino acid residues. A polypeptide is a long-chain of amino acid residues. A protein consists of one or more polypeptides which are folded in a globular or fibrous form, so that it performs a biological function.
Some of the foods tested will have a combination of peptides, polypeptides and protein and it is impossible to tell the relative concentration of each. Hence quantitative tests measure total protein.
FORMATION OF THE CHELATE COMPLEX
Lone electron pairs from 4 nitrogen atoms in the peptide bond coordinate a copper (II) ion.
A chelate complex is formed; the complex absorbs light at 540 nm and appears violet. Hence a color change from blue to violet indicates that proteins are present.
The greater the concentration of peptide bonds, the greater the color intensity. If the concentration of peptide bonds is low – such as when short-chain peptides are present - the color change is from blue to pink.
According to the Beer-Lambert Law, the absorption of the sample is directly proportional to the concentration of the species – in this case peptide bonds. Hence absorption spectroscopy using a spectrophotometer can be used to determine the concentration of total protein, following the biuret test.
As 2 peptide bonds are required for the formation of the chelate complex, single amino acids - no peptide bonds present - and dipeptides - only 1 peptide bond present – give a negative result.
Brilliant Biology Student 2013