Pressure-induced structural changes of pig heart lactic dehydrogenase

Lactic dehydrogenase from pig heart can be reversibly dissociated at hydrostatic pressures above 1000 bar. The breakdown of the native quaternary structure occurs at lower pressures compared to the isoenzyme from pig skeletal muscle. As shown by hybridization experiments of the two isoenzymes the final product of dissociation is the homogeneous monomer. Fluorescence emission spectra of the monomeric enzyme at elevated pressure are characterized by a decrease in fluorescence intensity without any red shift, indicating that no significant unfolding occurs upon high-pressure dissociation. The spectral changes are comparable to those observed after acid dissociation. The amount and rate of deactivation depend on pressure and on the conditions of the solvent. The presence of various anions (C1-, SO2-/4, HPO2-/4) has no effect on the stability of the enzyme towards pressure. High-pressure denaturation (as monitored by intrinsic protein fluorescence), and deactivation (measured immediately after decompression) run parallel; the pressure dependence of their first-order rate constants is characterized by an activation volume delta V not equal to De = - 140 +/- 10 cm3/mol. As taken from the yield of reconstitution, dissociation, denaturation and deactivation are found to be fully reversible provided the pressure does not exceed a limiting value (p = 1000 bar in Tris, pH 7.6; 24 h incubation at 20 degrees C). After extended incubation beyond the limiting pressure of 1000 bar, "irreversible high-pressure denaturation" occurs which is accompanied by partial aggregation after decompression. The coenzyme, NAD+, stabilizes the native tetramer shifting the dissociation equilibrium to higher pressures. The overall dissociation-association reaction can be quantitatively described by a consecutive dissociation/unfolding mechanism N in equilibrium 4 M' in equilibrium 4 M* (where N is the native tetramer, and M' and M* two different conformations of the monomer). The reaction volume of the dissociation reaction N in equilibrium 4 M' is found to be delta V Diss = - 360 +/- 30 cm3/mol; as indicated by the pressure dependence of the yield of reconstitution, the reaction volume of the equilibrium M' in equilibrium M* is also negative.