A probable mechanism of inactivation by urea of goat spleen cathepsin B. Unfolding and refolding studies.

Equilibrium and kinetic studies of the unfolding-refolding of goat spleen cathepsin B induced by urea are reported. Tryptophan fluorescence and enzyme activity were monitored. The activity of cathepsin B is lost reversibly at 1.2 M-urea. The enzyme unfolds in two main stages, having a stable intermediate (Y) between its native (N) and fully denatured (D) states. Enzyme activity and kinetic studies of these transitions indicate the existence of at least two intermediate forms (X1 and X2) between the N and Y states. The overall denaturation and renaturation scheme is thus suggested to be N in equilibrium with X1----X2 in equilibrium with Y in equilibrium with D. The multiplicity of the intermediate and fractional regaining of the activity up to a urea concentration of 2 M indicates the presence of multidomain structure in cathepsin B.