The solvent dependence of hydrogen exchange kinetics of folded proteins

The effects of ethanol, ethylene glycol, dioxane, and other organic cosolvents upon the hydrogen exchange rates of randomly coiled oxidized RNase, native RNase, and native trypsin were measured. The exchange rate of oxidized RNase, the model coupound for the proton transfer step in hydrogen exchange, is decreased by all of the cosolvents studied at temperatures in the range 3 to 20°. This was ascribed to the combined effects of the disruption of peptide bond solvation due to a reduction in the concentration of water and changes in [OH^-] ion concentration due to changes in the acid dissociation constant of water. The solvent dependence for both native RNase and native trypsin is similar in all of the solvents studied. At a low temperature (3 to 20°) the exchange rates go through a minimum as the solvent concentration is increased. At higher temperatures (20 to 35°) the exchange rates are increased at all concentrations of the cosolvent. The apparent rate minimum at lower temperatures is due to two opposing effects. Cosolvents decrease the rate of exchange that occurs directly from the folded molecule. At higher concentrations and higher temperatures, exchange rates are increased due to the increase in equilibrium concentration of unfolded protein resulting from the lowering of the thermal unfolding transition temperature. The decrease in rates for exchange directly from folded protein is primarily due to the effects on the proton transfer step, and not to binding or the solvent effects on protein structure. The solvents used in this study have no apparent effect on conformational processes contributing to the hydrogen exchange process in folded proteins.