Reconstitution of lactic dehydrogenase after acid dissociation. The yield of reactivation is determined by conformational rearrangements of the dissociated monomers.

Abstract

Reconstitution of lactic dehydrogenase from pig muscle after dissociation at acidic pH produces native tetramers and inactive aggregates, both of which have been previously described with respect to their structural properties and the kinetics of their formation. The ratio of the two fractions is determined by the relative rates of reassociation and aggregation [G. ZettlmeißI, R. Rudolph and R. Jaenicke (1979) Biochemistry 18, 5567–5571]. The present study is concerned with the influence of residual structural elements within the acid‐denatured monomers on the process of reconstitution.Long incubation at low pH leads to a marked decrease of the rate and yield of reactivation, caused by conformational changes within the partially unfolded monomers. These slow rearrangements are reflected by slight changes of the far‐ultraviolet circular dichroism of the acid‐dissociated monomers, indicating a decrease in helix content from 30 observed immediately after the transfer to low pH, to 25% after completion of the acid transition. The unchanged near‐ultraviolet circular dichroism and fluorescence properties prove local structural alterations to be of lesser importance compared to the changes of the residual backbone folding. Varying the experimental conditions during denaturation proves the slow decrease in the rate and yield of reactivation, as well as the changes in the far‐ultraviolet circular dichroism to be enhanced with incubation time and temperature (0–30°C). The first‐order rate constants of the decrease in the reactivation yield after incubation in 1 M glycine/H3PO4 pH 2.3, 0.1 M sodium phosphate pH 2.3, and 1 M H3PO4 were found to be in the same time range as the respective changes in the far‐ultraviolet circular dichroism.Apart from the similarity of the rate constants, the activation energy for both the acid induced changes of the yield of reactivation and the far‐ultraviolet circular dichroism is found to be identical (80 kJ/mol).The effects of low pH on the reconstitution of lactic dehydrogenase can be eliminated by complete disruption of the structure of the polypeptide chain, e.g. by adding 6 M guanidine · HCl. This proves unambiguously that the above‐mentioned structural rearrangements at acid pH are responsible for the observed decrease of the rate and yield of reactivation.