Biuret test
(Detection of peptide bond)
This is a general test for identification of proteins. This is positive for all compounds containing more than one peptide bonds.
Principle:
In alkaline medium, copper (II) sulphate (CuSO4) reacts with peptide bond nitrogen of peptides and proteins to form a violet coloured complex. The reaction is so named since this reaction is given by the substances biuret, which is obtained by condensation of 2 molecules of urea when heated to 180⁰C.
This reaction occurs only if the peptide molecule contains at least two peptide bonds (3 amino acids). Free amino acids and dipeptides do not undergo this test. The false-positive test result can also be observed when diamides of oxalic, malonic and succinic acid are present.
The biuret test generates complex compounds in which copper atom binds to nitrogen atoms of a peptide chain by the coordinate bond. The biuret test is used for the quantitative determination of high protein concentration in serum and other biological fluids as well. The determination of lower levels of proteins requires more sensitive methods.
Reaction:
Requirements:
- Protein solution:
- Preparation: Break egg shell, pour egg white in a beaker, add 100ml of distilled water, mix and filter through cotton. Use the filtrate for the test.
- 5% percent NaOH
- 1% glucose solution
- 5% copper sulphate
- Test tubes
- Pipettes
Procedure:
- To 2ml of each solution in different test tubes and add 2 ml of 5% percent NaOH on each.
- Then add 2 drops of 1% CuSO4 solution and mix.
- A violet color forms, which indicates the presence of peptide bonds in the molecule.
Image Source: https://www.getmygrades.co.uk/subjects/biology-gcse-ib/2-1-5-1biuret/
Result Interpretation:
Image Source: http://biocheminology.blogspot.com/2015/03/test-for-protein.html
- Positive test: Proteins are not present if there is no color change (i.e. solution remains blue )
- Negative test: Protein or peptides are present if the solution turns from blue to violet (i.e. deep purple)
Note:
- Addition of excess amount of copper sulphate should be avoided, otherwise blue color will develop instead of violet color.
- Biuret reaction is used in the quantitative estimation of proteins in biological fluids.
References:
- http://biocheminology.blogspot.com/2015/03/test-for-protein.html
- https://www.getmygrades.co.uk/subjects/biology-gcse-ib/2-1-5-1biuret/
- https://www.chemistrylearner.com/biuret-test.html
- https://onlinesciencenotes.com/biuret-test-principle-requirements-procedure-and-result-interpretation/
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