Techniques to Monitor Disulfide Bond Formation and the Reduction Potential of Cysteine-Cystine Couples In vitro and In vivo

Among many biological reduction-oxidation (redox) couples, the cysteine-cystine couple (where cystine denotes two disulfide-linked cysteines) is of central importance for cellular processes. It can serve both in low molecular weight diffusible redox cofactors and in conserved reactive motifs that are integrated into peptides and proteins. Protein-bound cysteine-cystine couples exhibit highly context-specific thermodynamic and kinetic features and serve a large variety of structural and functional purposes. This chapter reviews the methodological repertoire to monitor the redox state and specific reactivity of biological cysteine pairs. Determination of thermodynamic parameters such as the standard reduction potential of cysteine-cystine couples or the pKa of individual cysteines requires classical biochemical approaches using purified components. In their native environment, however, redox state analyses of cysteine pairs are complicated by ex vivo artifacts, which must be minimized using appropriate thiol quenching methods. Special attention is given to fluorescent protein-based real-time sensors. These reporter proteins can be applied to virtually any living specimen to monitor biological redox data with ample spatial and temporal resolution and, in some cases, high specificity for a single redox couple.