Abstract

Protein cysteine sulfhydryl groups are susceptible to a number of redox-dependent modifications, including an interchange between the reduced sulfhydryl and an oxidized disulfide state. A growing body of evidence suggests that reversible disulfide bond formation alters the structure and function of proteins. In this chapter, a method is described for isolating disulfide bonded proteins from different subcellular compartments by using a differential detergent fractionation technique followed by sequential nonreduc-ing/reducing two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (i.e., Redox 2D-PAGE). This method can be adapted to examine individual redox-active proteins by immunoprecipi-tating an epitope-tagged redox protein expressed in cultured cells and using Redox 2D-PAGE and mass spectrometry to identify proteins that form mixed disulfides with the tagged protein. With the use of these techniques, it is shown that disulfide bond formation occurs within families of cytoplasmic proteins and may provide a common mechanism used to control multiple physiological processes.

Key words: Redox signaling, disulfide bonding, protein folding, two-dimensional electrophoresis, mass spectrometry, epitope-tagging.

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