Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation

The colored complex formed between Cu + and bicinchoninic acid is the basis of the bicinchoninic acid protein assay (P. K. Smith, R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C. Klenk (1985) Anal. Biochem. 150, 76–85). Studies show that cysteine, cystine, tryptophan, tyrosine, and the peptide bond are capable of reducing Cu 2+ to Cu +. Electrochemical studies and the magnitude of the color changes observed when the reaction is carried out at 37°C indicate that tryptophan, tyrosine, and the peptide bond are not completely oxidized at this temperature. When the reaction temperature is increased to 60°C, significantly more color formation is observed for these three groups. Studies with di-, tri-, and tetrapeptides and with proteins indicate that the extent of color formation is not the sum of the contributions of the individual color producing functional groups. Compounds with functional groups similar to those of cysteine, cystine, tyrosine, or tryptophan are shown to react with the bicinchoninic acid reagent. The color formed by these compounds in the presence of bovine serum albumin cannot be compensated for by using a reagent blank containing an identical concentration of the interfering compound.