Unfolding and refolding of phospholipase C from Bacillus cereus in solutions of guanidinium chloride

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Abstract
Protein-fluorescence studies indicated that phospholipase C from B. cereus is denatured in solutions of guanidinium chloride. The denaturation was not thermodynamically reversible and followed biphasic kinetics. Guanidinium chloride solutions released the structural Zn2+ from the enzyme and rendered all histidine residues chemically reactive. In the presence of free Zn2+ the enzyme was much more resistant to denaturation. Also, the addition of free Zn2+ to the denatured enzyme induced refolding. The Zn2+-free apoenzyme was much more sensitive to guanidinium chloride than was the native enzyme and the denaturation appeared to be thermodynamically reversible. Guanidinium chloride denaturation was associated with a reversible inactivation of the enzyme. Heat-inactivated, coagulated enzyme was substantially re-activated on dissolution in guanidinium chloride solutions followed by dialysis against a Zn2+-containing buffer.