When considering protein folding with a transient intermediate, a difficulty arises as to determination of the rates of separate transitions. Here we overcome this problem, using the kinetic studies of the unfolding/refolding reactions of the three-state protein apomyoglobin as a model. Amplitudes of the protein refolding kinetic burst phase corresponding to the transition from the unfolded (U) to intermediate (I) state, that occurs prior to the native state (N) formation, allow us to estimate relative populations of the rapidly converting states at various final urea concentrations. On the basis of these proportions, a complicated experimental chevron plot has been deconvolved into the urea-dependent rates of the I$N and U$N transitions to give the dependence of free energies of the main transition state and of all three (N, I, and U) stable states on urea concentration.Keywords: protein folding; folding intermediates; tryptophan fluorescence; chevron plot; stoppedflow; apomyoglobin Apomyoglobin is a good object for protein folding studies because its thermodynamic (Griko et al. 1988;Hughson et al. 1990;Jennings and Wright 1993;Jamin et al. 2000) and structural (Barrick and Baldwin 1993a,b;Eliezer and Wright 1996;Eliezer et al. 1998;Jamin and Baldwin 1998;Jamin et al. 1999;Lecomte et al. 1999;Tcherkasskaya and Ptitsyn 1999;Tsui et al. 1999;Tcherkasskaya et al. 2000) properties in both the native and intermediate conformational states are well elucidated. At neutral pH, apomyoglobin structure is ''native'' globular, with seven of eight helices of holomyoglobin tightly packed (A-E and G, H; while F, involved in the heme binding, is disordered in apoprotein) (Eliezer and Wright 1996). At pH 4.2, the native structure undergoes the transition into a thermodynamically stable ''molten globule' ' (Dolgikh et al. 1981; Ptitsyn 1995) intermediate state (Griko et al. 1988) that contains three helices (Hughson et al. 1990), A, G, and H. This intermediate has two sub-states (stable at pH 4.2 and pH 3.9), which convert one into the other within a millisecond time range (Jamin and Baldwin 1998;Jamin et al. 1999). It was shown using the stopped-flow and quenchflow techniques that urea-induced apomyoglobin refolding goes via a kinetic intermediate that forms within 6 msec and is structurally similar to the equilibrium molten globule intermediate observed at pH 4.2 (Jennings and Wright 1993). Subsequent kinetic studies suggested that this intermediate is on-pathway (Jamin and Baldwin 1998). Quenchflow amide proton exchange combined with mass-spectrometry confirmed that apomyoglobin folds by a single pathway and that the intermediate is obligatory (Tsui et al. 1999). NMR analysis of mutant apomyoglobins also showed that some point mutation may change the folding pathway of apomyoglobin (Garcia et al. 2000).The presence of a kinetic intermediate on the apomyoglobin folding pathway makes this protein attractive for studies of three-state folding/unfolding reactions. Protein folding involves a transition state and, for the m...

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