Studies on Protein Subunits

Abstract

Abstract The renaturation of guanidinium chloride-denatured aldolase in buffer containing 20% sucrose at 0° can be resolved into two distinct processes by following the regain of activity. Initially, an active intermediate is formed which is unstable to treatment with 2.3 m urea. This intermediate is then converted into another active species which is stable to 2.3 m urea. Analysis of the kinetic data shows that the over-all process of reactivation is independent of the concentration of enzyme in the renaturation mixture whereas the rate of formation of 2.3 m urea-stable activity is highly concentration-dependent. The results are consistent with a model in which denatured aldolase refolds in a first order reaction to give active monomers which then associate in concentration-dependent steps to give active dimer, trimer, and tetramer. In this model, the activity of each subunit is the same whether it exists singly or as part of an oligomer. However, only the tetramer is stable to 2.3 m urea. This model is also supported by gel filtration studies of partially renatured aldolase. The results support our earlier observation based on matrix-bound aldolase subunits (Chan, W. W.-C. (1970) Biochem. Biophys. Res. Commun., 41, 1198) that aldolase subunits are active but differ from the tetramer in being unstable toward 2.3 m urea.