Modeling of interaction of basic lipophilic ligands with cytochrome-p-450 reconstituted in liposomes - determination of membrane partition-coefficients of s-(-)-nicotine and n,n-diethylaniline from spectral binding-studies and fluorescence quenching
Biochimica et Biophysica Acta vol:1107 issue:1 pages:93-104
The spectral interaction of N,N-diethylaniline and S-(-)-nicotine with cytochrome P450IIB4 reconstituted into large unilamellar vesicles could properly be described by a model for interaction of basic lipid-soluble ligands with membrane-bound acceptor sites in which linear partitioning of non-ionized ligand in the membrane is postulated. Apparent spectral dissociation constants K(s)app for type I binding of N,N-diethylaniline and for type II binding of S-(-)-nicotine increased linearly with increasing lipid volume fraction alpha-L of the proteoliposomes. From plots of K(s)app vs. alpha-L, the membrane partition coefficient of each ligand was calculated. The apparent affinity of cytochrome P450IIB4 for the ligands increased as the pH was raised from 6.0 to 8.5. However, effective dissociation constants were virtually independent of the pH, indicating that only the uncharged form of the basic ligands interact with cytochrome P450IIB4. For each compound, the apparent quenching rate constants k(q)app derived from the Stern-Volmer plots for dynamic quenching of the fluorescence intensity of 8-(2-anthryl)octanoic acid in liposomes, decreased with increasing liposomal concentration. Plots of (k(q)app)-1 vs. alpha-L Of the liposomes yielded the overall bimolecular quenching rate constant k(q) of each quencher. The k(q) value for S-(-)-nicotine was about three orders of magnitude less than that for N,N-diethylaniline. The values of the partition coefficient of N,N-diethylaniline, obtained from the binding studies and the fluorescence quenching measurements, were identical (on average, K(p) amounted to 383). Analysis of the quenching data of N,N-diethylaniline with Scatchard plots likewise revealed that the association of the compound with liposomal membranes is a pure partition process.