Biuret Reaction

 Proteins form biuret-like compounds (structure I) in alkaline solution by losing a proton from the nitrogen atoms of the peptide bonds. In strong alkaline solution and Cu²⁺, this compound forms a blue colored complex (structure II). This color reaction is called the biuret reaction. The sensitivity of the reaction is not very great, but the color yield is similar from protein to protein. Thus, this reaction has been employed to quantify proteins in widely varying samples. 

According to the method of Gornall et al. (1), 1 to 10 mg of protein is determined by absorbance at 540 nm after incubation with biuret reagent (CuSO₄, potassium sodium tartrate, NaOH) for 30 min at 20° to 25°C. An improved biuret method by Westley and Lambeth (2), which removes the free Cu²⁺ ion with an ion-exchange resin and adds Na-diethyldithiocarbonate as a coloring reagent, detects 0.05 to 1 mg of protein. A simple and sensitive micro-biuret method was developed by Itzhaki and Gill (3), where UV absorbance at 310 nm is employed to quantify protein.

References

1. A. G. Gornall, C. S. Bardawill, and M. M. David (1949) J. Biol. Chem. 177, 751–766.

2. J. Westley and J. Lambeth (1960) Biochim. Biophys. Acta 40, 364–366. 

3. R. F. Itzhaki and D. M. Gill (1964) Anal. Biochem, 9, 401–410.