Formation of a Purple Product upon the Reaction of ABTS Radicals with Proteins

The reaction of the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) free radical (ABTS(●)) with proteins (bovine serum albumin, blood plasma, egg white, erythrocyte membranes, and Bacto Peptone) leads not only to a reduction of ABTS(●) but also to the appearance of a purple color (absorption maximum at 550–560 nm). The aim of this study was to characterize the formation and explain the nature of the product responsible for the appearance of this color. The purple color co-precipitated with protein, and was diminished by reducing agents. A similar color was generated by tyrosine upon reaction with ABTS(●). The most feasible explanation for the color formation is the addiction of ABTS(●) to proteins’ tyrosine residues. The product formation was decreased by nitration of the bovine serum albumin (BSA) tyrosine residues. The formation of the purple product of tyrosine was optimal at pH 6.5. A decrease in pH induced a bathochromic shift of the spectra of the product. The product was not a free radical, as demonstrated by electrom paramagnetic resonance (EPR) spectroscopy. Another byproduct of the reaction of ABTS(●) with tyrosine and proteins was dityrosine. These byproducts can contribute to the non-stoichiometry of the antioxidant assays with ABTS(●). The formation of the purple ABTS adduct may be a useful index of radical addition reactions of protein tyrosine residues.