36] Measuring thiol-disulfide exchange equilibriumconstants for single cysteine-containing proteins

Abstract

Publisher Summary This chapter describes the single-step disulfide exchange reaction involving protein thiols and discusses the measurement of thiol–disulfide exchange equilibrium constants for single cysteine-containing proteins. The mechanism of thiol–disulfide exchange involves attacking a thiolate anion on the disulfide in a single concerted step—that is, an S N 2 mechanism. In the transition state, significant negative charge builds up on the attacking and leaving sulfur atoms, as well as on the central sulfur atom. Because the thiolate is the active species, the reaction kinetics is greatly affected by the pH and the p K a of the attacking thiol. The equilibrium constants for disulfide exchange are highly pH dependent if there is a difference in the p K a values of the attacking and leaving thiols. Because disulfide exchange is often coupled to protein activity and stability, much useful information may be obtained from measurements of thiol–disulfide exchange reactions involving proteins. This method has the advantage over standard chemical modification and mutagenesis procedures in that the disulfide exchange reaction is a reversible process and therefore can be studied and quantitated thermodynamically.