Kinetics of (-)¹²⁵Iodocyanopindolol Binding to Intact Human Mononuclear Cells

Abstract

In association experiments of (-)¹²⁵Iodocyanopindolol (¹²⁵ICYP) with human mononuclear cells (MNC) at 70 pM and a temperature of 37°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, K_d, 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, R_o, decreased from 970 ± 170 to 713 ± 58 sites/cell. Analysis of computer simulated binding curves confirmed the decrease in K_d and R_o 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 K_d- and to a lesser extent of R_o-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 R_o.