Hydrostatic pressure and calcium-induced dissociation of calpains.

Abstract The dissociation of μ- and m-calpains was studied by fluorescence spectroscopy under high hydrostatic pressure (up to 650 MPa). Increasing pressure induced a red shift of the tryptophan fluorescence of the calcium-free enzyme. The concentration dependence of the spectral transition was consistent with a pressure-induced dissociation of the subunits. Rising temperature increased the stability of calpain heterodimers and confirmed the predominance of hydrophobic interactions between monomers. At saturating calcium, the spectral transition was not observed for native or iodoacetamide-inactivated calpains, indicating that they were already dissociated by calcium. The reaction volume was about −150 ml mol −1 for both isoforms, and the dissociation constants at atmospheric pressure are approximately 10 −12 M and 10 −15 M for μ- and m-calpains, respectively. This result indicates a tighter interaction in the isoform that requires higher calcium concentration for activity.