title: pH-induced folding/unfolding of staphylococcal nuclease: determination of kinetic parameters by the sequential-jump method.
abstract: 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 in equilibrium with D1 in equilibrium with D2 in equilibrium with 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.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, University of Minnesota College of Biological Sciences, St. Paul 55108.
authors.forename: H M
authors.initials: HM
authors.lastname: Chen
authors.forename: V S
authors.initials: VS
authors.lastname: Markin
authors.forename: T Y
authors.initials: TY
authors.lastname: Tsong
mesh_terms: D004795:Enzyme Stability; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D008836:Micrococcal Nuclease