RECONSTITUTION OF LACTIC-DEHYDROGENASE - NON-COVALENT AGGREGATION VS REACTIVATION .1. PHYSICAL-PROPERTIES AND KINETICS OF AGGREGATION

The reconstitution of lactic dehydrogenase after dissociation in 6 M guanidine hydrochloride or at acidic pH leads back to active tetramers indistinguishable from the native enzyme. In addition, aggregates of “irreversibly denatured” enzyme are formed to a certain extent. It has been shown that the ratio of both fractions depends on such factors as the extent of denaturation and the concentration of the enzyme in the process of reconstitution. The electron microscopical analysis of the aggregates shows a broad distribution of high molecular weight particles (Mr<106). As suggested by circular di-chroism measurements in the far-UV and by degradation in strong denaturants, these particles are composed of individual monomeric chains with partially restored secondary structure. The aggregates are stabilized by noncovalent interactions. For determination of the factors responsible for the competition of reactivation and aggregate formation, the kinetics of aggregation were analyzed by stopped-flow laser light scattering experiments and compared with the kinetics of reactivation [cf. Rudolph, R., & Jaenicke, R. (1976) Eur. J. Biochem. 63, 409-417]. Aggregation is determined by a process with a reaction order greater than 2, competing with a fast first-order folding reaction in the pathway of reactivation. Reactivation and aggregate formation seem to be kinetically controlled; the ratio of both fractions depends solely on the kinetics of formation and not on the relative conformational energies of the native enzyme and its aggregates. A general model for the competition of reactivation and aggregation is proposed, including the influence of denaturing and labilizing solvent conditions. © 1979, American Chemical Society. All rights reserved.