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 (U-B 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 U-B state is accompanied by an unusual expansion of the chain involving an intermediate, I-alc, 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 I-alc state are generally constrained relative to those in the U-B state. A few nuclear magnetic resonance-detected tertiary interactions are also found in the I-alc state. The fact that the I-alc 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 U-B state indicate a definite influence of electrostatic repulsion severed by the low dielectric of the water/alcohol mixture. Interestingly, the U-B reversible arrow I-alc segment of the U-B (sic) I-alc (sic) 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 submilllisecond hydrophobic collapse during which almost the entire buried surface area associated with the U-B state is recovered, suggesting the overwhelming influence of hydrophobic interaction over electrostatic repulsions.