B04 Protein Assay by Reduction of Copper(II)

Description
Bicinchoninic acid solution and Cu(II) sulfate are mixed to form a green solution. Protein reduces the green Cu(II) bicinchoninic acid complex, whose stoichiometry has not been studied, to a violet Cu(I) bidentate complex. A set of five standards at different protein concentrations is shown. A saliva sample is tested and compared to these standards. The protein reduction of copper(II) to copper(I) is complex. Approximately 50% of the reaction is due to reducing amino acids like cysteine(A26), and the other 50% is due to the reduction by the peptide linkage of the protein.

Equation
Cu(II)(Bicinchoninate)_x + protein --> Cu(I)(Bicinchoninate)₂³^-

Reference
Pierce Catalog 1989, p. 202.

P. K. Smith et al. Anal. Biochem 150, 76 (1985).

J. Peisach, P. Aisen, and W. E. Blumberg, Eds, "The Biochemistry of Copper", Academic Press, (1966) p. 18.