A Kinetic Approach to Characterize the Electrostatic Environments of Thiol Groups in Proteins

In this study, we synthesized a zwitterionic DTNB derivative, 5-(2-aminoethyl)-dithio-2-nitrobenzoate (ADNB), and characterized its reactions with several cationic, anionic, and neutral thiols. Reactions with ADNB, unlike those with DTNB, are relatively insensitive to electrostatic environments and ionic strengths. At relatively low ionic strength, rate ratios,kADNB/kDTNB, varied from 0.22 for reactions with low-molecular-weight cationic thiols to 3.0 for those with low-molecular-weight anionic thiols. AkADNB/kDTNBratio of ∼200 for Cys-34 of BSA appears to reflect a very anionic environment.kADNB/kDTNBratios of ∼6 and ∼1, respectively, for canine and equine serum albumins, which have Glu-82 → Asp and Glu-82 → Ala substitutions suggest Glu-82 is the most important anionic residues affecting the reactivity of Cys-34 in BSA.kADNB/kDTNBratios appear to be useful for characterizing electrostatic environments of thiol groups in proteins.