Abstract
We have used the method of modulated excitation (Ferrone, F.A., and J.J. Hopfield, 1976, Proc. Natl. Acad. Sci. USA. 73:4497-4501), with an improved apparatus and a revised analytical procedure, to measure the rate of conformational change between the oxy (R) and deoxy (T) conformations of triligated carboxy-hemoglobin A at pH 6.5 and 7.0. We have found the rates to be kRT = 1.2 X 10(3) s-1 and kTR = 3.5 X 10(3) s-1 for pH 6.5, while for pH 7.0, kRT = 1.0 X 10(3) s-1, and kTR = 3.0 X 10(3) s-1. The value for L3, the equilibrium constant between conformations, was virtually unchanged between pH 6.5 and 7.0. While the rates measured here differ from those obtained in the original use of this method, these new rates are fully consistent with the original data when analyzed by the revised procedures presented here. When taken with other kinetic and equilibrium data, our measurements suggest that the transition state between structures is dominated by the behavior of the T quaternary structure. Finally, a spectral feature near the HbCO Soret peak has been observed that we ascribe to an allosteric perturbation of the spectra of the liganded hemes.