An analysis of the stopped-flow kinetics of gaseous ligand uptake and release by adult mouse erythrocytes

Abstract

Ligand uptake and release by the haemoglobin contained within adult mouse erythrocytes was studied by using dual-wavelength stopped-flow techniques. The rate of O2 uptake is very much lower than that expected for an equivalent concentration of haemoglobin in free solution. The O2-concentration-dependence found in uptake experiments is greater than first-order. CO uptake shows the same pattern of reactivity as does O2, but the associated rates of uptake are lower and the concentration-dependence of the CO rates is first-order. O2 release from the adult erythrocytes was measured by stopped-flow mixing with Na2S2O4. Under these circumstances the deoxygenation of intracellular haemoglobin shows accelerating time courses. The apparent rate-constant-dependence on dithionite concentration shows a rate limit at high reductant concentrations. Computer simulations of both ligand uptake and release processes were carried out by using a three-dimensional model. The simulations clearly indicate that in rapid-mixing experiments the rather slow experimentally observed O2 uptake rate is due to rate-limiting diffusion through an extracellular stagnant solvent layer. In the case of O2 release, however, the major rate-controlling process is the rate of O2 dissociation from the haemoglobin molecules, which accelerates during the deoxygenation process.