DETERMINATION OF THE CARBON-MONOXIDE BINDING CONSTANTS OF MYOGLOBIN MUTANTS - COMPARISON OF KINETIC AND EQUILIBRIUM METHODS

The carbon monoxide (CO) binding constants of human myoglobin (Mb) and several single-site mutants have been determined using two different methods. In the kinetic method, which is commonly used for this ligand , the overall association (k(on)) and dissociation (k(off)) rates of C O were measured by flash photolysis and NO replacement, respectively, and the ratio k(on)/k(off) was calculated. In the equilibrium method, the binding constant K-eq was measured directly using a thin-layer tec hnique. These two measurements yield similar results for human wild-ty pe Mb but differ significantly for some of the mutants. Possible reaso ns for these discrepancies are analyzed. A model assuming the presence of interconverting conformers with different association and dissocia tion rates is considered in light of infrared measurements on the CO s tretching frequency in the MbCO forms of the same proteins [Balasubram anian et al. (1993a) Proc. Natl. Acad. Sci. U.S.A. 90, 4718]. It is su ggested that in the case of some mutants which exhibit multiple confor mations, this model may lead to nonequilibrium kinetics, which could p roduce the observed discrepancies between the kinetic and equilibrium determinations of the binding constant. These results suggest that bot h equilibrium and kinetic data should be obtained, even for a monomeri c protein such as Mb, before the relative stabilities of mutants can b e meaningfully compared.