Photolytic studies on the carbon monoxide complex of sulphaemoglobin.

The CO complex of sulphaemoglobin was found to be photolytically dissociable at pH 6.0 and pH 8.5. The recombination kinetics of the CO complex after flash photolysis show two phases, which differ in rate by approximately 20-fold. At both pH 6.0 and pH 8.5 the two phases show an identical linear dependence on CO concentration, with associated second-order rate constants of 6.0 x 10(3) M-1 . s-1 and 1.2 x 10(5) M-1 . s-1 respectively. The percentage contribution of each of the two phases to the total absorbance change is independent of the CO concentration employed, but is a function of the protein concentration used. The percentage of the faster phase increases with protein dilution. These results taken together with computer simulation suggest the existence of appreciable amounts of monomeric species in solution at both pH 6.0 and pH 8.5 at low concentration of liganded protein. Estimates of the dissociation constants for the dimer in equilibrium monomer equilibrium yield values of 8 microM and 3.5 microM at pH 6.0 and pH 8.5 respectively. Stopped-flow delayed-flash-photolysis studies show that the liganded dimer decays to the liganded monomer with a half-life of approx. 0.5 s. Constant-illumination experiments allow an estimation of the half-life for the combination of deoxy monomers to deoxy dimers of 22 s. A model explaining the range of CO recombination kinetics seen for sulphaemoglobin does not require the existence of a photolytically produced high-activity form, even at high pH, in contrast with the case for normal haemoglobin.