Kinetics of formation of the 4,4′-bis-dimethylaminodiphenyl carbonium ion (BDC +) and its reaction with sulfhydryl residues

Abstract

The use of 4,4′-bis-dimethylaminodiphenylcarbinol (BDC-OH) as an analytical reagent for sulfhydryl residues and as a specific chemical modification reagent for proteins is dependent upon the unique properties of the BDC + cation present in aqueous buffers below a pH of 6.5. In the presence of aqueous buffers, pH 5.1, BDC + exhibits a λ max of 606 nm with an apparent molar absorption coefficient of 10,000 m −1 cm −1. Upon the addition of 4 m guanidine hydrochloride this apparent coefficient is enhanced to 70,800 m −1 cm −1. The true molar extinction coefficient for BDC + was determined to be 128,000 m −1 cm −1. The reaction of BDC + with sulfhydryl residues of proteins or simple thiols is rapid and leads to a complex devoid of visible color. In the pH range 3.0–7.0, a complex equilibrium is established among the three species BDC-OH, BDC +, BDCH ++. The formation of this equilibrium is proton mediated, and is discussed in terms of the equilibrium, rate, and acid dissociation constants.