Binding of two spin-labelled derivatives of chlorpromazine to human erythrocytes.

The binding to human intact erythrocytes of two different spin-labelled derivatives of chlorpromazine has been studied. The influence of the positively charged side chain of the drug has been the focus of our attention. The positively charged amphiphilic compound (spin derivative I) is water-soluble up to 80 microM at pH values below 5.9. The apolar analogue (spin derivative II) aggregates in aqueous buffer from the lowest concentration tested. Both spin derivatives undergo a slow reduction inside the erythrocyte. The reduced nitroxides are readily reoxidized by adding a low, non-quenching, concentration of potassium ferricyanide to the intact erythrocytes. The fractions of spin label I and II bound to the erythrocyte membrane or to the erythrocyte-extracted lipids remain constant as a function of the temperature (3-42 degrees C) and as a function of the concentration of the spin label up to 150 microM. E.s.r. spectra of both spin labels show a two-component lineshape when they are bound to intact erythrocytes. Below 35 degrees C for the positively charged spin probe, and below 32 degrees C for the apolar spin probe, the simulation of the lineshape shows that more than 50% of the spectrum originates from a slow-motion component. This slow-motion component is also found in erythrocyte-extracted lipids probed by the positively charged spin label below 25 degrees C. In contrast, no slow-motion component is detected in the range 4-40 degrees C for the apolar spin label in erythrocyte-extracted lipids. In this environment the apolar probe experiences a single fast anisotropic motion with an exponential dependence on 1/temperature. Detailed lineshape simulations take into account the exchange frequency between binding sites where the probe experiences a fast motion and binding sites where it experiences a slow motion. The exchange frequency is strongly temperature-dependent. Characterization of the different motions experienced inside the different locations has been achieved and compared for whole erythrocytes and for the extracted lipids. The biochemical nature of the binding sites (membrane protein/acidic phospholipid) giving rise to the slow-motion component is discussed as a function of the polarity of the spin-labelled drug and as a function of the temperature controlling the fluidity of the lipid bulk and influencing the distribution of the drug inside the membrane.