ALKALINE DENATURATION AND PARTIAL REFOLDING OF PEPSIN INVESTIGATED WITH DAPI AS AN EXTRINSIC PROBE

The binding parameters of DAPI to porcine stomach pepsin have been des cribed in the previous article in this issue (A. Mazzini et al.). Here we exploit the differences in the spectroscopic (fluorescence and cir cular dichroism) properties of DAPI bound to either native or alkali d enatured pepsin. We follow the kinetics of pepsin denaturation around neutrality (pH range 6.8-7.4), at several phosphate buffer ionic stren gths (range 0.02-0.25). The dependence of the apparent dissociation ra te constant on pH clearly shows that the rate limiting step follows th e dissociation of about three acidic protein residues, The acceleratin g effect by ionic strength we observed can be accounted for by a simpl e treatment based on both transition state theory and Debye-Hueckel's limiting law. Furthermore, when a solution of pepsin, rapidly denature d at pH 7, is reacidified to a pH between 4.5 and 5.5, a substantial r ecovery of protein secondary structure, with no enzymatic activity, is observed, judging by the far UV circular dichroism of the protein. Th is process of partial refolding can easily be followed using DAPI as a n extrinsic reporter group, able to monitor the kinetics of formation and decay of a highly fluorescent intermediate. This process becomes f aster at a lower pH, at least in the limited range investigated (pH 4. 5-5.5), in which the refolded protein does not aggregate, but, in cont rast to unfolding, is almost independent in ionic strength. (C) 1997 E lsevier Science B.V.