STUDY OF DEOXYHEMOGLOBIN BY LASER PHOTOLYSIS

Laser photolysis experiments were carried out at 20(DEGREES)C and pH 8.3 on carboxyhemoglobin in three viscous solution systems to determine the effect of viscosity on the rate K(,o) of the R(,o) (--->) T(,o) conformational change. These rates were determined by fitting the Two State model (Monod et al J. Mol. Biol. 12: 88-118) to CO recombination data. For all three solvent systems K(,o) decreased with increasing viscosity in the general manner predicted by Gavish (Biophys. Struct. Mech. 4: 37-52) who suggested that viscous damping reduces the amplitude of structural fluctuations leading to conformation change. For sucrose/borate solution systems K(,o) decreased from 6400 S('-1) to 1500 S('-1) as viscosity was increased from 1.0 to 5.94 cP.; A new laser photolysis method was used to measure K(,42)('T(,o)) the tetramerdimer dissociation constant for unliganded hemoglobin in the pH range 9.5 to 11.2. Modeling of the hemoglobin concentration dependence of the CO rebinding observed following laser photolysis of hemoglobin samples with a few percent bound CO allowed the determination of K(,42)('T(,o)). K(,42)('T(,o)) was found to increase monotonically from 0.037 to 640 (mu)M over the pH range from 9.5 to 11.2. This new method produces K(,42)('T(,o)) values which are about an order of magnitude more precise than those determined previously.; Studies of the reaction of the first CO molecule with homoglobin were performed at 20(DEGREES)C in 0.1M phosphate buffer at pH 7.0 using flow/laser photolysis. This reaction is found to be much more complex than previously thought. In particular a conformational change to a rapidly reacting state appears to occur within the single CO bound subunit in an Hb(CO)(,1) molecule. This change is complete within about 20 seconds after the CO is bound and leaves the other three deoxysubunits in their normal slowly reacting state with a CO binding rate constant 0.10 (mu)M('-1) S('-1) while the CO bound heme converts to a state where it has a binding rate constant 60 times larger. This observation contradicts the basic assumption of the Two State model that the binding properties of all subunits change in a concerted fashion.