Kinetics of refolding and reactivation of rabbit-muscle aldolase after acid dissociation.

Tetrameric rabbit muscle aldolase has been dissociated to the monomer at pH 2.3 and fully reassociated and reactivated at pH 7.6 Kinetics of reactivation and refolding were followed by slow and fast kinetic techniques after neutralization of the acidic enzyme solutions by dilution or rapid mixing. Rate constants (k), reaction orders (n), and activation evergies (E) were calculated from measurements on the time, concentration, and temperature dependence of the reactions. The experimental results prove reactivation at high enzyme concentration (c greater than 4mug/ml) to obey first-order kinetics; at lower concentrations a transition to a higher reaction order is observed. Because of concentration-dependent deactivation at low enzyme concentrations reactivation measurements could not be extended below c approximately o.5 mug/ml. In the accessible concentration range incomplete separation of reassociation and transconformation processes as well as intrinsic residual activity of the isolated subunits lead to an average value of n = 1.40 +/- 0.18. Renaturation as monitored by protein fluorescnce is a multi-step process composed of a fast increase in flurescence emission (first-order rate constant k approximately 15 s-1) and a slow concentration-dependent decrease which parallels the recovery of enzyme activity (n = 1.46 +/- 0.12). The activation energy of both processes is of the order of E = 12-16 Kcal/mol (50-67 kJ/mol). Reassociation is a prerequisite of full catalytic function and native fluorescence.