Effect of glycerol on the pressure dissociation of tetrameric LDH using high-pressure native PAGE velocity method

We used the high-pressure native PAGE velocity method to investigate the effect of glycerol on the dissociation process of pig heart L-lactate dehydrogenase (LDH), a tetrameric protein. High pressure is known to disrupt oligomerization without significantly affecting the folding of individual monomers. Our methodology allowed us to evaluate the complete dissociation process of LDH, distinguishing between the tetramer-to-dimer and dimer-to-monomer steps, as well as aggregation. Adding 10% glycerol was found to affect the dimer-to-monomer step, shifting the dissociation pressure toward higher values. In contrast, the dissociation pressure for the tetramer-to-dimer step appeared unaffected. However, a more detailed analysis of the pressure dependence of the equilibrium constant revealed that glycerol stabilized the tetramer state relative to the dimer state by approximately 3 kJ/mol at ambient pressure and that this stabilizing effect declined as the pressure increased, shifting toward destabilization above 40 MPa. We interpreted these cosolvent effects in terms of the preferential binding to the newly exposed inter-subunit interface on the dissociation.