Kinetics of (-)125Iodocyanopindolol binding to intact human mononuclear cells

In association experiments of (-)125Iodocyanopindolol (125ICYP) with human mononuclear cells (MNC) at 70 pM and a temperature of 37 degrees C equilibrium was reached within 30 min. However, when the same experiments were performed at a concentration of 4 pM 125ICYP, equilibrium was only reached after 3 hours. The consequences of incomplete equilibrium for the interpretation of binding experiments under the incorrect assumption that equilibrium has been reached, was investigated at equilibration times of one, two and three hours. The dissociation constant, Kd, decreased from 7.4 +/- 0.2 pM after one hour to 2.5 +/- 0.4 pM after three hours of incubation while the receptor density, RO, decreased from 970 +/- 170 to 713 +/- 58 sites/cell. Analysis of computer simulated binding curves confirmed the decrease in Kd and RO at prolonged incubations. We conclude that in 125ICYP binding in intact MNC one hour of incubation is not sufficient to obtain equilibrium at the lower concentrations. This leads to an overestimation of Kd- and to a lesser extent of RO-values. Extending the incubation time to three hours on the other hand may lead to a loss of cells and therefore to an underestimation of RO