SOLVENT ISOTOPE EFFECTS ON THE REFOLDING KINETICS OF HEN EGG-WHITE LYSOZYME

Solvent isotope effects have been observed on the in vitro refolding k inetics of a protein, hen lysozyme. The rates of two distinct phases o f refolding resolved by intrinsic fluorescence have been found to be a ltered, to differing extents, in D2O compared with H2O, and experiment s have been conducted aimed at assessing the contributions to these ef fects from various possible sources. The rates were found to be essent ially independent of whether backbone amide nitrogens were protiated o r deuterated, indicating that making and breaking of their hydrogen bo nding interactions is not associated with a substantial isotope effect . Neither were the rates significantly affected by adding moderate con centrations of sucrose or glycerol to the refolding buffer, suggesting that viscosity differences between H2O and D2O are also unlikely to e xplain the isotope effects. The data suggest that different factors, a cting in opposing directions, may be dominant under different conditio ns. Thus, the isotope effect on the rate-determining step was found to be qualitatively reversed on going to low pH, suggesting that one com ponent is probably associated with changes in the environments of carb oxylate groups in forming the folding transition state. This term disa ppears at low pH as these groups are protonated and an opposing effect then dominates. It was not possible to identify this other effect on the basis of the present data, but a dependence of the hydrophobic int eraction on solvent isotopic composition is a likely candidate.