Asynchronous ligand binding and proton release in a root effect hemoglobin.

CO binding to the Root effect hemoglobin of menhaden, Brevoortia tyrannus, has been studied by flash photolysis and equilibrium measurements in [bis(2-hydroxyethyl)amino]Tris(hydroxymethyl)methane and Tris buffers, containing 0.2 M NaCl, between pH 6.0 and 8.0. The equilibrium and kinetic data were analyzed according to the two-state model, extended to include chain differences. The calculated value of the allosteric constant, L, varied from 3 X 10(6) at pH 6.0 to 20 at pH 8.0, lower at each pH value than that computed for phosphate buffer. In addition, the intrinsic rate constants of both T and R states were found to vary with pH. The kinetics of CO binding and of proton release, followed by absorbance changes in the pH indicator dye phenol red, were observed in 0.2 M NaCl, at pH values ranging from 6.3 to 7.8. Proton release lags behind CO binding across this pH range, the larger lags occurring at lower pH; this suggests that some proton release is associated with quaternary conformational change. The CO binding progress curves in unbuffered solution were simulated by the two-state model; in these calculations the value of L was systematically changed during the course of the reaction. The time courses of reaction intermediates, obtained from these computations, were then used to represent the kinetics of proton release. A simple model, assuming that proton release accompanies quaternary conformational transition but a modified model, incorporating pH dependence of the intrinsic T and R state affinities, describes proton release across the pH range studied.