Determining the Redox Potential of a Protein Disulphide Bond

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