A method for estimating free Ca within human red blood cells, with an application to the study of their Ca-dependent...

Murphy, Coll, Rich and Williamson (J. Biol. Chem. 255:6600--6608, 1980) described a null-point method for estimating intracellular free Ca in liver cells. They used digitonin to lyse the cells in solutions of varying Ca concentration. This method has been adapted for use with human red cells. The values found are about 0.4 micron or micrometer Ca in fresh cells, and from 0.4 to 0.7 micron or micrometer Ca in blood-bank cells, at pH 7.2 and 37 degrees C. These are likely to be overestimates, and the errors and limitations of the method are discussed. Red cells may be loaded with Ca by metabolic depletion in Ca-containing solutions. Such cells have an elevated K permeability, and the relationships between free Ca, total Ca and K permeability were investigated, using 86Rb as a tracer for K. 86Rb flux studies show that the affinity of the K channel for Ca is the same in cells as in resealed ghosts where intracellular Ca can be controlled with Ca buffers, but the rate of tracer equilibration is 3-6 times faster in ghosts than in cells.