Unfolding Action of Alcohols on a Highly Negatively Charged State of Cytochrome c

It is well-known that hydrophobic effect play a major role in alcohol–protein interactions leading to structure unfolding. Studies with extremely alkaline cytochrome c (UB state, pH 13) in the presence of the first four alkyl alcohols suggests that the hydrophobic effect persistently overrides even though the protein carries a net charge of −17 under these conditions. Equilibrium unfolding of the UB state is accompanied by an unusual expansion of the chain involving an intermediate, Ialc, from which water is preferentially excluded, the extent of water exclusion being greater with the hydrocarbon content of the alcohol. The mobility and environmental averaging of side chains in the Ialc state are generally constrained relative to those in the UB state. A few nuclear magnetic resonance-detected tertiary interactions are also found in the Ialc state. The fact that the Ialc state populates at low concentrations of methanol and ethanol and the fact that the extent of chain expansion in this state approaches that of the UB state indicate a definite influence of electrostatic repulsion severed by the low dielectric of the water/alcohol mixture. Interestingly, the UB ⇌ Ialc segment of the UB ⇌ Ialc ⇌ U equilibrium, where U is the unfolded state, accounts for roughly 85% of the total number of water molecules preferentially excluded in unfolding. Stopped-flow refolding results report on a submillisecond hydrophobic collapse during which almost the entire buried surface area associated with the UB state is recovered, suggesting the overwhelming influence of hydrophobic interaction over electrostatic repulsions.