The biuret reaction can be used to estimate the amount of protein in a sample. Like biuret, proteins contain a
-CONH- group which, when reacted with aqueous alkaline solution of copper(II) sulphate, produces the following complex of a blue-violet colour:
The violet color produced, allows the estimation of protein colorimetrically. The colour intensity is proportional to the amount of peptide bonds participating in the reaction. A series of samples are prepared with known amounts of protein along with a sample with the unknown amount of protein. Samples are then transferred to cuvettes and the absorbances measured. the absobance of the unknown sample should fall between the absorbances of the known sample.The complex has an absorbance maximum of 540 nm. A calibration curve can then be produced and the unknown sample's concentration calculated using Beer's Law.
The Cu(II)SO4 is added to the protein dissolved in sodium hydroxide. The sodium hydroxide breaks up the secondary sructure of the protein, i.e., the alpha and beta pleated sheets so it is easier for the Cu2+ ions to access and form complexes with the nitrogen atoms in the proteins.
One disadvantage of this reaction, however, is that some of the Cu2+ will react with NaOH to produce the insoluble, Cu(OH)2