Ionic effects on self-reactivation of p-hydroxymercuribenzoate-inhibited lactate dehydrogenase

Abstract

During the course of an investigation of ionic effects on the structure and recombination of beef heart lactate dehydrogenase (BHLDH), it was observed that, following treatment with the sulfhydryl reagent, p-hydroxymercuribenzoate (PMB), to reduced enzyme activity by 90%, the enzyme would spontaneously reactivate when placed in neutral 0.1 m sodium phosphate buffer. As much as 87% of the enzyme activity was restored by this process. During self-reactivation the quantity of PMB bound to the enzyme (i.e., moles of inhibitor per mole of enzyme) remains essentially constant. The rate and magnitude of self-reactivation for enzyme which has been inactivated in the presence of phosphate ions differ in different ionic environments. In the presence of 0.1 m arsenate, the rate and magnitude of self-reactivation are identical to those attained in phosphate buffer. However, relatively little self-reactivation occurs when PMB-inactivated enzyme is incubated in neutral 0.1 m Tris-HCl. In the presence of nitrate and borate ions no reactivation occurs and residual activity of the inhibited enzyme is rapidly destroyed. From these studies on PMB inhibition and binding to LDH it appears that, in different ionic environments, different—SH groups become available for interaction with sulfhydryl reagents. Moreover, the effects of different ions on enzyme activity, inhibition, and recovery indicate that LDH exists in several different enzymically active conformations.