Abstract
The alkaline-induced unfolding and the salt-induced refolding of high molecular mass and low molecular mass goat brain cystatins under high pH conditions have been monitored by enzyme inhibitory activity, intrinsic and extrinsic fluorescence, and CD measurements. An increase in pH to 14.0 at low ionic strength was accompanied by complete loss of the activity of both cystatins. The maximum activity for HMGBC was observed at pH 8.0 whereas pH 7.0 was pH optimum for LMGBC. The results obtained for HMGBC indicate that, at low ionic strength, an increase in pH enhanced the extent of unfolding of the enzyme to the maximum unfolded state at pH 14.0. The unfolding occurred via a simple native to unfolded state transition. As for LMGBC, at low ionic concentration a pH increase from 7 to 10 caused unfolding of the cystatin, while at pH 12, we observed the formation of a compact intermediate state. Thus, the alkaline-induced LMGBC unfolding occurs via three step transition from native to intermediate, and, then, to unfolded state. At pH 12, an increase in the ionic strength by addition of salts (KCl/Na2SO4) resulted in the folding of both alkaline-denatured cystatins. The salt-induced refolded inhibitors (HMGBC/LMGBC) also showed significantly higher activity compared to the unfolded forms in presence of salts. Na2SO4 was observed to be more efficient in refolding the alkaline-unfolded cystatins at low concentrations (1 M) as compared to the KCl (3 M).
The above results suggest that the salt-induced folding high pH is governed by an intermediate state which may also exists during the folding process of several other proteins.