Thiol/disulfide exchange and redox potentials of proteins

The sulfhydryl group of the amino acid cysteine plays a pivotal role in the structure and function of numerous proteins. The high nucleophilicity of the thiolate anion coupled with its reasonably low pKa produces high chemical reactivity in nucleophilic additions and substitutions, accounting for its wide-spread use as a catalytic group in a variety of enzymes catalyzing hydrolysis, substitution, and exchange reactions. In addition, the reversible two electron oxidation converting the thiol of cysteine to its disulfide form is used not only in electron transfer reactions but also in crosslinking and stabilizing the tertiary and quarternary structure of proteins, the regulation of enzyme activity, and the protection of the intracellular environment. The direct application of electrochemical methods to the study of the redox properties of thiols, either in small molecules or in proteins, is complicated by interactions of the thiol with most electrode surfaces. Consequently, there have been few reliable electrochemical measurements of the redox potentials of cysteine and other biological thiols and disulfides. Nevertheless, alternate methodology involving reversible thiol/disulfide exchange equilibria has allowed the estimation of standard redox potentials of the sulfhydryl groups of biologically important thiols, including those found in proteins. In this chapter, the redox chemistry and biochemistry of the sulfhydryl groups of proteins will be outlined, methodology for applying the thiol/disulfide exchange reaction to the measurement of redox potentials will be described, and examples of the biological function of these redox state changes will be given.