Multiple parameter kinetic studies of the oxidative folding of reduced lysozyme.

Abstract

We have compared the oxidative renaturation of reduced hen egg white lysozyme promoted by Cu(II) + O2 with that promoted by a glutathione redox buffer. The progress curves for protein fluorescence, circular dicroism, thiol oxidation, hydrodynamic volume, and enzymic activity were determined for both regeneration systems. All of these processes were more rapid in the glutathione regeneration than in the copper-catalyzed. Comparison of the two systems was carried out by normalizing the progress curves with a coordinate system where "time" is replaced by "extent of protein thiol oxidation." While similar progress curves were obtained for circular dichroism, the two systems produced distinctly different progress curves for enzymic activity, fluorescence, and gel permeation chromatographic reflection of protein hydrodynamic volume. We infer that all these differences result from differences in relative amounts and/or kind of reaction intermediates. Thus, there are substantial differences between the renaturation mechanisms of the glutathione- and the copper-promoted systems.