Kinetics of reassociation and reactivation of pig-muscle lactic dehydrogenase after acid dissociation.

Lactic dehydrogenase from pig skeletal muscle (M4) can be reversibly dissociated to the monomer at pH 4-5 depending on the anion applied. Using identical experimental conditions the pH-depending profiles of dissociation, denaturation, and deactivation coincide with each other. Deviations in the pH-dependence of protein fluorescence reflect changes in the microenvironment of specific chromophores rather than significant differences in the structure-function relationship as pH is changed. The dissociation state is characterized by the homogeneous inactive monomer of 35000. The reassociated material consists of up to 85% fully active tetramers, indistinguishable from the initial native enzymes, as shown by hydrodynamic, spectroscopic and enzymic properties. The rest represents a mixture of irreversibly denatured high aggregates. Under optimum conditions of reactivation both recovery of enzymic activity and native fluorescence obey strict second order kinetics with an activation energy of 44 kcal/mol (184 kJ/mol). NAD+ and NADH do not show any significant influence on the yield and kinetics of the refolding process and the recovery of enzymic activity. The kinetic results suggest the reassociation of inactive monomers to be rate-limiting in the refolding and reactivation processes being considered.