A method for studying oxygen diffusion barrier in erythrocytes: effects of haemoglobin content and membrane cholesterol.

1. In order to study the kinetics os the oxygen egress from human red cells in the 50 sec-20 min time range, an apparatus for measuring the oxygen dissociation process was constructed, combining a spectrophotometer with an oxygen electrode of quick response. 2. Starting from air-saturated haemolysate or red cell suspensions, the velocity of oxygen dissociation from oxyhaemoglobin (Vdiss) and of oxygen disappearance in the medium (Vobs) after addition of bakers' yeast (consuming the dissolved oxygen at the velocity of Vconsump) were recorded. A parameter (r) was defined as the ratio of two velocities, Vegress (the velocity of oxygen egress into the medium) and Vdiss, r identical to Vegress/Vdiss = (Vconsump -Vobs)/Vdiss. Vcomsump could be calculated by the Michaelis-Menten equation as follows, Vconsump = Vmax [O2]/(Km + [O2]), where Vmax was the maximal velocity of oxygen consumption of bakers' yeast. 3. The r value was always 1.0 for the haemolysate, but it was less than 1.0 for the normal red cells. Further, the oxygen dissociation curve of red cells obtained at higher Vmax was distorted, due to the non-equilibration between intra- and extracellular oxygen concentrations. 4. The r value was (i) independent of the amounts of the allosteric effectors (2,3-diphosphoglycerate and H+) but (ii) dependent on the haemoglobin contents and (iii) dependent on the amounts of the membrane cholesterol. Therefore, the r value reflected only the process of the oxygen diffusion but not the "chemical reaction' rate. The "barrier' of the oxygen diffusion decreased at lower haemoglobin contents, but increased at higher cholesterol contents in the membrane.