Biuret Reaction - Protein in Egg Albumen

Demonstration Experiment on Video

Biuret Reaction - Protein in Egg Albumen

Objective: Test for Peptides and Proteins

Peter Keusch

German version

Chemicals and material:
1 M NaOH
1 M aqueous copper(II) sulfate
glycine
egg albumen

Glass wares:
3 conical measures, graduated, 500 mL
1 beaker 500 mL
3 beakers 50 mL
measuring cylinder 100 mL
2 volumetric pipets 5 mL
1 volumetric pipet 10 mL
4 volumetric pipets 20 mL
5 snap-cap vials 20 mL
pipet ball<
3 glass stirring rods

Hazards and safety precautions:

Sodium hydroxide solution is very corrosive. Causes severe burns. May cause serious permanent eye damage. Very harmful by ingestion.

Safety glasses, protective gloves!

Experimental procedure:

Preparation of the egg albumen "solution": The albumem of an egg is mixed with 300 mL aqueous NaCl.

Three conical measures are set up as described in the following table. Each of the mixtures is mixed with a glass stirring rod.

conical measure 1	400 mL of water	-	5 mL of 1 M copper sulfate solution	5 mL of 1 M NaOH
conical measure 2	400 mL of water	one spatula of glycine	5 mL of 1 M copper sulfate solution	5 mL of 1 M NaOH
conical measure 3	400 mL of water	30 mL egg albumen "solution"	20 mL of 1 M copper sulfate solution	40 mL of 1 M NaOH

Results:

conical measure 1	water	light blue precipitate
conical measure 2	glycin	blue solution
conical measure 3	egg albumen	blueviolet solution

Video clip (Download RealPlayer .rm file)

Discussion:

Peptides and proteins (long-chain polypeptides) react with Cu ²⁺ in alkalinity to create a blueviolet colored chelate complex with an absorbance maximum at 540 nm (conical measure 3). The peptide must have three peptide bonds at least.

Amino acids and Cu ²⁺ ions form a blue complex (conical measure 2).

The biuret test is used for the quantitative photometrical determination of total protein concentration. The intensity of the color produced in the biuret reaction is proportional to the number of peptide bonds participating in the reaction.

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