Reassociation of lactic dehydrogenase from pig heart studied by cross-linking with glutaraldehyde

Cross-lining with glutaraldehyde has been successfully applied in order to analyze the kinetics of reassociation of oligomeric enzymes (R. Hermann, R. Rudolph, and R. Jaenicke Nature 277, 243 - 245 (1979). In the present study other assembly of lactic dehydrogenase from pig heart is investigated using this approach. In order to eliminate pertubations caused by excessive folding reactions, acid dissociation was performed in the presence o 0.8 M Na2SO4 at 0 degrees C. Under optimum conditions complete cross-linking during reconstitution proves the dimer to be the only intermediate of reassociation. The dimer leads to tetramer transition is found to be rate-limiting for both reassociation and reactivation, suggesting the tetramer to be the enzymatically active species. The presence of monomers during reconstitution indicates that tetramer formation is preceded by a fast monomer-dimer equilibrium. The kinetics model describing the experimental data.