Determination of the partition of the tetramisole derivative (+/-)-5,6-dihydro-6-phenyl-2-normal-propyl-imidazo[2,1-b]thiazole into liposomal membranes by fluorescence quenching of the membrane probe 8-(2-anthryl)-octanoic acid
European biophysics journal with biophysics letters vol:21 issue:1 pages:47-56
Fluorescence quenching has been used to study the partition of the tetramisole derivative (+/-)-5,6-dihydro-6-phenyl-2-n-propyl-imidazo[2,1-b]thiazole into liposomes, consisting of a mixture of egg L-alpha-phosphatidylcholine, egg phosphatidylethanolamine and dipalmitoylphosphatidic acid (2:1:0.06 w/w/w). The tetramisole derivative quenched the fluorescence of the intramembrane probe 8-(2-anthryl)-octanoic acid. The quenching process could be rationalized by a model for dynamic quenching in which an intermediate excited-state non-emitting complex (PQ)* between neutral quencher (Q) and excited probe (P*) is involved: Q(A) half-arrow-right-over-half-arrow-left Q(L) + P(L)* k(-d) half-arrow-right-over-half-arrow-left k(d) (PQ)L* --> k(i) P + Q where k(d), k(-d) and k(i) represent the rate constants of complex formation, dissociation and deactivation, respectively. The subscripts A and L denote the aqueous and lipid phases, and the asterisk indicates the excited state. Linear Stern-Volmer plots were obtained from quenching experiments of fluorescence intensities and fluorescence lifetimes. The slopes of the plots were dependent on the lipid volume fraction of the liposomes. Measurement of the reciprocal of the apparent bimolecular quenching rate constant at various lipid volume fractions yielded the partition coefficient K(p) and the overall quenching rate constant k(q)[k(q) = k(d)k(i)/(k(i) + k(-d)] of the tetramisole derivative. The steady-state measurements were performed at three different pH-values. Time-correlated single photon counting measurements revealed a single-exponential fluorescence decay for 8-(2-anthryl)-octanoic acid in the presence and absence of quencher. The quenching results were in accordance with the model that only the neutral form is capable of partitioning into the lipid phase. Combined average values of 318 and 6.59 x 10(8) M-1 s-1 were calculated for the partition coefficient and the bimolecular quenching rate constant, respectively, from the steady-state and time-resolved quenching experiments.