Determining the Redox Potential of a Protein Disulfide Bond.

The redox potential of a protein disulfide bond is one of the most important factors for determining the role of a disulfide bond. Disulfide bonds could have a stabilizing role for the structure of a protein or they can play a functional role that can regulate protein activity or binding. Determining the redox potential of disulfides can help distinguish the functional from the structural disulfide bonds. In this chapter, two methods for determining the redox potential of a protein disulfide bond are described. The first method uses maleimide-biotin labeling of free cysteine thiols and western blot densitometry to determine the fraction of reduced disulfide bond under various redox buffering conditions. The second method uses differential cysteine labeling and tandem mass spectrometry to determine the redox potential.