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 02 uptake is very much lower than that expected for an equivalent concentration of haemoglobin in free solution. The 02-concentration-dependence found in uptake experiments is greater than first-order. CO uptake shows the same pattern of reactivity as does 029 but the associated rates of uptake are lower and the concentration-dependence of the CO rates is firstorder. 02 release from the adult erythrocytes was measured by stopped-flow mixing with Na2S204. 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 02 uptake rate is due to rate-limiting diffusion through an extracellular stagnant solvent layer. In the case of 02 release, however, the major rate-controlling process is the rate of 02 dissociation from the haemoglobin molecules, which accelerates during the deoxygenation process.

Stopped-flow kinetics of oxygen uptake and release by embryonic red blood cells

“…Dithionite solutions were prepared according to the procedures of Coin & Olson (1979) and the final solutions were calibrated for activity by titration against standard ferricyanide solutions (Creutz & Sutin, 1973;Brittain & Simpson, 1989).…”

“…Dual-wavelength stopped-flow was performed as described previously (Brittain & Simpson, 1989). In stopped-flow studies of embryonic cells, the degree of cell lysis produced by shear forces in the stopped-flow apparatus was approx.…”

“…Data capture and analysis were performed as previously outlined (Brittain & Simpson, 1989), with the exception that in a few experiments where the sample was very limited, such that a final haemoglobin concentration of only 1.5 /uM was used, Fourier filtering of the reaction traces was employed to remove the noise generated mainly by the optical chopper of the dualwavelength stopped-flow apparatus . 02 equilibrium measurements were made using the thin film techniques previously described (Lapennas et al, 1981;Wells, 1982).…”

“…where Do (1.2 x l0-5cm2 s-1) and DHbO (1.9x l0-7 cm2 s 1) are the diffusion coefficients for 02 and oxyhaemoglobin and r is the radial distance from the centre of the cell (account being taken of the volume excluded by the cell nucleus). As three distinct compartments exist within this model, namely the bulk solvent, stagnant layer and cell cytoplasm, similar equations must be derived for each compartment (Vandergriff, 1984;Brittain & Simpson, 1989). Account must also be taken of the fact that the hydrodynamics of the stopped-flow apparatus are such that the thickness of the stagnant layer surrounding the red blood cell grows with time after flow stops.…”

“…Over the past sixty years, since the pioneering studies of Hartridge & Roughton (1927), many studies have been made of 02 uptake and release of whole red blood cells with the aim of establishing a working model of red blood cell function, culminating in the three-dimensional disc model of Vandergriff (1984). This model has been employed to accurately describe 02 uptake and release from a range of adult red blood cell types (Vandergriff, 1984;Vandergriff & Olson, 1984a,b,c;Brittain & Simpson, 1989). However, this model has not previously been developed to describe ligand-exchange processes in any other than biconcave disc-shaped erythrocytes.…”