Submillisecond unfolding kinetics of apomyoglobin and its pH 4 intermediate

Submillisecond mixing experiments and tryptophan fluorescence spectroscopy are used to address two questions raised in earlier stopped-flow studies of the folding and unfolding kinetics of sperm whale apomyoglobin. A study of the pH 4 folding intermediate (I) revealed, surprisingly, that its folding and unfolding kinetics are measurable and fit the two-state model except f or a possible burst phase in unfolding. Submillisecond mixing experiments c onfirm the unfolding burst phase and show that its properties are consisten t with the recently discovered interconversion between two forms of I, Ia r eversible arrow Ib. In urea-induced unfolding, Ib is converted to Ia before Ia unfolds, and the unfolding kinetics of Ia fit the two-state model when the burst phase is assigned to Ib --> Ia. The second question is whether th e Ia, Ib intermediates accumulate transiently when the native protein (N) u nfolds to the acid unfolded form (U). Earlier work showed that Ia and Ib ac cumulate when U refolds to N at PH 6.0 and the results fit the linear foldi ng pathway U reversible arrow Ia reversible arrow Ib reversible arrow N. We report here that either or both Ia and Ib accumulate transiently when N un folds to U at PH 2.7 and that the position of the rate-limiting step in the pathway changes between unfolding at pH 2.7 and refolding at pH 6.0. In un folding as in refolding, we do not detect a fast track that bypasses the Ia , Ib intermediates. (C) 1999 Academic Press.