Disulfide bond within µ-calpain active site inhibits activity and autolysis

Oxidative processes have the ability to influence µ-calpain activity. In the present study the influence of oxidation on activity and autolysis of µ-calpain was examined. Furthermore, LC-MS/MS analysis was employed to identify and characterize protein modifications caused by oxidation. The results revealed that the activity of µ-calpain is diminished by oxidation with H2O2 in a reversible manner involving cysteine and that the rate of autolysis of µ-calpain concomitantly slowed. The LC-MS/MS analysis of the oxidized µ-calpain revealed that the amino acid residues 105–133 contained a disulfide bond between Cys(108) and Cys(115). The finding that the active site cysteine in µ-calpain is able to form a disulfide bond has, to our knowledge, not been reported before. This could be part of a unique oxidation mechanism for µ-calpain. The results also showed that the formation of the disulfide bond is limited in the control (no oxidant added), and further limited in a concentration-dependent manner when β-mercaptoethanol is added. However, the disulfide bond is still present to some extent in all conditions indicating that the active site cysteine is potentially highly susceptible to the formation of this intramolecular disulfide bond.