title: Solvent isotope effects on the refolding kinetics of hen egg-white lysozyme.
abstract: Solvent isotope effects have been observed on the in vitro refolding kinetics of a protein, hen lysozyme. The rates of two distinct phases of refolding resolved by intrinsic fluorescence have been found to be altered, to differing extents, in D2O compared with H2O, and experiments have been conducted aimed at assessing the contributions to these effects from various possible sources. The rates were found to be essentially independent of whether backbone amide nitrogens were protiated or deuterated, indicating that making and breaking of their hydrogen bonding interactions is not associated with a substantial isotope effect. Neither were the rates significantly affected by adding moderate concentrations of sucrose or glycerol to the refolding buffer, suggesting that viscosity differences between H2O and D2O are also unlikely to explain the isotope effects. The data suggest that different factors, acting in opposing directions, may be dominant under different conditions. Thus, the isotope effect on the rate-determining step was found to be qualitatively reversed on going to low pH, suggesting that one component is probably associated with changes in the environments of carboxylate groups in forming the folding transition state. This term disappears 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 interaction on solvent isotopic composition is a likely candidate.
authors.affiliation: Department of Biochemistry, University of Cambridge, United Kingdom.
authors.forename: L S
authors.initials: LS
authors.lastname: Itzhaki
authors.forename: P A
authors.initials: PA
authors.lastname: Evans
mesh_terms: D000818:Animals; D002645:Chickens; D004529:Egg White; D006863:Hydrogen-Ion Concentration; D007554:Isotopes; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry*; D017510:Protein Folding; D012997:Solvents / Q000737:chemistry*; D014783:Viscosity
chemical_list: D007554:Isotopes; D012997:Solvents; D009113:Muramidase