Laser Photolysis Studies of Oxy- and Carbonylhemoglobin in Red Blood Cells. Effects of Cell Membrane on Reversible Binding of O2 and CO

Nanosecond laser photolysis studies of hemoglobin in red blood cells (RBCs) were performed to elucidate the effects of cell membrane on the oxygen uptake and release mechanism of RBCs. Oxy RBCs readily photodissociated O2 by the 355-nm laser pulse to give deoxy RBCs, which return to oxy RBCs at 1 atm air within ca. 50 μs after the pulse. The decay of deoxy RBCs is expressed as a sum of the two exponential functions of time, indicating that the oxygen-rebinding reaction is composed of the two processes. The rate constants, k f and k s, for the faster and slower processes of the oxygen rebinding, measured as a function of the oxygen concentration, revealed that (1) k f is independent of the oxygen concentration, [O2], and (2) k s asymptotically increases with an increase in [O2] to a limiting value. These kinetic results were markedly different from those obtained with the laser photolysis of oxyhemoglobin molecules in aqueous solutions. On the basis of the kinetic studies for oxygen rebinding, the cell membrane of RBCs is suggested to dominate the rates for oxygen uptake and release of hemoglobin in RBCs. For comparison with oxygen, the CO-binding reactions of deoxy RBCs and deoxyhemoglobin molecules in aqueous solutions were studied by laser flash photolysis. The decay profiles of deoxy RBCs produced by laser photolysis of carbonyl RBCs at various CO concentrations were identical with those of deoxyhemoglobin molecules in aqueous solutions. Thus, in contrast to O2, CO molecules are supposed to freely diffuse in and out of RBCs through the cell membrane.