Refolding and unfolding kinetics of the equilibrium folding intermediate of apomyoglobin.

Little is known about the kinetic process in which stable intermediates in protein folding are formed: whether their folding is highly cooperative (two-state) or weakly cooperative is controversial. We report here that the folding and unfolding kinetics of the pH 4-stable intermediate (I1) of apomyoglobin are measurable, in the millisecond time range, when monitored by stopped-flow measurements of tryptophan fluorescence. The kinetics confirm that folding of I1 is strongly cooperative, but there is a burst phase (missing amplitude) in unfolding. If the faster steps in unfolding of I1 can be measured directly by suitable fast-reaction methods, they will give information about the nature of the folding transition.