Clinical Chemistry, Vol 33, 481-485, Copyright © 1987 by American Association for Clinical Chemistry

Temperature-dependent binding of cyclosporine to an erythrocyte protein

RP Agarwal, GA Threatte and RA McPherson

In this competitive binding assay to measure endogenous binding capacity for cyclosporine (CsA) in erythrocyte lysates, a fixed amount of [3H]CsA plus various concentrations of unlabeled CsA is incubated with aliquots of a test hemolysate. Free CsA is then adsorbed onto charcoal and removed by centrifugation; CsA complexed with a cyclosporine-binding protein (CsBP) remains in the supernate. We confirmed the validity of this charcoal-separation mode of binding analysis by comparison with equilibrium dialysis. Scatchard plot analysis of the results at 4 degrees C yielded a straight line with slope corresponding to a binding constant of 1.9 X 10(7) L/mol and a saturation capacity of approximately 4 mumol per liter of packed erythrocytes. Similar analysis of binding data at 24 degrees C and 37 degrees C showed that the binding constant decreased with increasing temperature, but the saturation capacity did not change. CsBP was not membrane bound but appeared to be freely distributed within erythrocytes. 125I-labeled CsA did not complex with the erythrocyte CsBP. Several antibiotics and other drugs did not inhibit binding between CsA and CsBP. These findings may explain the temperature- dependent uptake of CsA by erythrocytes in whole blood and suggest that measurement of CsBP in erythrocytes or lymphocytes may help predict therapeutic response or toxicity after administration of CsA.