Biuret Test For Protein - Brilliant Biology Student

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Biuret Test for Proteins

Picture Benedict's Test for Non-reducing Sugar Picture Ethanol Emulsion Test for Lipids Picture Iodine Test for Starch Picture Benedict's Test for Reducing Sugar

What is the Biuret Test for Proteins?

The Biuret Test is often used to determine the presence of peptide bonds in protein. As a student you will most likely will be testing for the presence of protein in foods. The Biuret test for proteins may also be extended to quantitatively measure the concentration of total protein using spectrometric methods.The Biuret reagent is the sole reagent in the Biuret Test for proteins. The Biuret reagent contains
  • Hydrated Copper sulphate
  • Potassium hydroxide solution
  • Potassium sodium tartrate

Are the alternative reagents for the Biuret Test for Proteins?

As an alternative to the Biuret Reagent, either of the following solutions may be used to yield comparable results.
  • Sodium Hydroxide and Copper Sulphate Solutions
  • Fehling’s Solutions A and B

PROCEDURE

Three procedures are outlined below using different reagents.For each of the procedures, a liquid sample must be prepared from solid foods as follows.PREPARATION OF LIQUID SAMPLECrush 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.

What is the procedure for the Biuret Test for proteins using Biuret Reagent?

Add 2 cm3 of the liquid food sample* to a clean, dry test tube
  • Add 2 cm3 of Biuret Reagent.
  • Repeat steps the steps above with de-ionized water to prepare a negative control and with albumin (egg white) to prepare a positive control.
  • Shake well and allow the mixture to stand for 5 minutes
  • Observe any color change.

What is the procedure for the Biuret Test for protein using sodium hydroxide and copper sulphate solutions?

  • Add 1 cm3 of sodium hydroxide solution (40% or bench solution) and 1% copper (II) sulphate solution dropwise (one drop at a time) to the food sample
  • Repeat the steps above with de-ionized water to prepare a negative control and with albumin (egg white) to prepare a positive control.
  • Shake well and allow the mixture to stand for 5 minutes
  • Observe any color change.

What is the procedure for the Biuret Test for protein using Fehling's A and B Solutions?

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.
  • Repeat steps 1 and 2 with de-ionized water to prepare a negative control and with albumin (egg white) to prepare a positive control.
  • Shake well and allow the mixture to stand for 5 minutes
  • Observe any color change.

OBSERVATIONS & INTERPRETATION

What are the expected results in the Biuret Test for proteins?

Picture Look for colour changes in the solution. They range from no colour change (blue) to pink to deep violet.Colour changes are best visualized against a white background such as a white tile or a sheet of paper.
OBSERVATIONSNo change (solution remains blue )The solution turns from blue to violet (deep purple)The solution turns from blue to pink INTERPRETATIONProteins are not presentProteins are presentPeptides are present (Peptides or peptones are short chains of amino acid residues)

DISCUSSIONQUESTIONS & ANSWERS

Who or what is Biuret?

The biuret reagent is not named after z famous scientis but after a substance called biuret (H2NC(O)NHC(O)NH). Biuret is 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.

What is the composition of the Biuret reagent?

Biuret Reagent contains
  • Hydrated Copper sulphate. This provides the Cu (II) ions which form the chelate complex. Cu (II) ions give the reagent its characteristic blue color.
  • Potassium hydroxide solution does not participate in the reaction but provides the alkaline medium.
  • Potassium sodium tartrate (KNaC4H4O6·4H2O) – stabilizes the chelate complex.

What is the principle - or basis - of the Biuret Test for proteins?

Picture 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.Lone electron pairs from 4 nitrogen atoms in the peptide bond coordinate a copper (II) ionto form the chelate complex. The chelate complex absorbs light at 540 nm so 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.

Why would amino acids give a negative result in the Biuret Test for Proteins?

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.

How can the Biuret Test be extended to quantitatively measure the concentration of protein?

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 is a quantitative method which can be used to determine the concentration of total protein, following the Biuret test.
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