On the basis of previous stopped-flow pH-jump experiments, we have proposed that the acid-and alkaline-induced folding/unfolding transition of staphylococcal nuclease, in the time range 2 ms to 300 s, follows the pathway N0 = D1 = D2 = D3, in which D1, D2, and D3 are three substates of the unfolded state and N0 is the native state. The stopped-flow “double-jump” technique has been employed to test this mechanism and to determine the rate constants which would not be accessible by the direct pH jump because of the lack of fluorescence signal, i.e., the rates for the conversion of D1 to D2 and of D2 to D3. In the forward jump, a protein solution kept at pH 7.0 was mixed with an acidic or alkaline solution to the final pH of 3.0 or 12.2, respectively. The mixed solution was kept for varying periods of time, called the delay time, tD. A second mixing (the back jump) was launched to bring the protein solution back to pH 7.0. The time course of the Trp-140 fluorescence signals recovered in the back jump was analyzed as a function of tD. Kinetics of the unfolding were found to be triphasic by the double-jump method, contrary to the monophasic kinetics observed by the direct pH jump. Complex kinetics of unfolding are expected with the proposed kinetic scheme. Analysis of data obtained by both the direct-jump and the double-jump experiments yielded complete sets of rate constants and activation energies for the unfolding to pH 3.0 and to pH 12.2 and for the folding from acid and alkali to pH 7.0. Fractions of protein in N0, Db D2, and D3 states were determined to be 1.0, 0, 0, and 0, respectively, at pH 7.0; 0, 0.61, 0.28, and 0.11, respectively, at pH 3.0; and 0, 0.43, 0.30, and 0.27, respectively, at pH 12.2. ÅG of the transition between any two neighbors of the three D's was less than 0.55 kcal mol−1. Thus, any obligatory pathway in an early stage of the chain folding must be determined by the kinetic barriers rather than by the stability of the intermediate states.
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