Effects of D2O on the Association-Dissociation Equilibrium in Subunit Proteins

Heavy water has been used as a tool to study the role of water in the association-dissociation equilibrium of subunit proteins. In the case of lactate dehydrogenase, D2O was found to stabilize the enzyme at low ionic strength and inhibit subunit interchange between heart- and muscle-type enzyme in solution. When frozen, however, subunit interchange occurred to a similar extent in either D2O or H2O. In the case of bovine liver glutamate dehydrogenase, D2O appears to facilitate the association of monomers to form polymers. The maximum weight average molecular weights of glutamate dehydrogenase were found to be approximately doubled in D2O compared with H2O, and the dissociation constant of the polymer appeared to be much smaller in D2O. The weight average molecular weights were calculated from sedimentation velocity and diffusion rate measurements in the analytical ultracentrifuge. Because these measurements were made at 8°, at which the contribution of hydrophobic bonding would be minimal, it was concluded that the stabilizing action of D2O reflected the increased strength of deuterium bonds and deuterium water bridges. Alteration of the association-dissociation equilibrium in regulatory proteins by D2O is a possible mechanism to explain certain of the potent inhibitory effects of D2O on biological systems.