Kinetic analysis of the inhibition of sulfate transport in human red blood cells by isothiocyanates

Summary

A kinetic analysis of anion self-exchange in human red blood cells, in the presence of an irreversible inhibitor, is presented and applied to the study of the inactivation of sulfate transport by three isothiocyanates: 3-isothiocyano-1,5-naphthalenedisulfonic acid, disodium salt (INDS), 1-isothiocyano-4-naphthalene sulfonic acid, sodium salt, monohydrate (INS), and 1-isothiocyano-4-benzenesulfonic acid, sodium salt, monohydrate (IBS). The time dependence of the inhibition of sulfate transport by the isothiocyanates used could be described by a single exponential and could be shown to contain a reversible and an irreversible component. In each case a portion of sulfate efflux was found to be resistant to inactivation. The residual portion of the sulfate efflux varied with inhibition: 4% for INS, 16% for INDS, and 34% for IBS. INS showed the largest reversible inhibitory effect (12% of the flux remaining at 0.2mm inhibitor concentration), while INDS showed the weakest effect (92% of the flux remaining at 0.3mm inhibitor concentration). IBS had the highest rate of inactivation while INDS had the lowest. The kinetic analysis further suggests that all three isothiocyanates bind reversibly to an inhibitory site on the membrane before they bind covalently, and therefore irreversibly, to the same site on the membrane. The equilibrium constant for the dissociation of the reversibly-bound complex,K _i, and the rate of irreversible inactivation after all membrane sites are reversibly bound,k _max, have been computed for all three inhibitors: INDS (K _i=420μm,k _max=5.04 hr⁻¹), INS (K _i=148 μm,k _max=6.48 hr⁻¹), and IBS (K _i=208 μm,k _max=8.11 hr⁻¹).