Dynamic fluorescence quenching studies on lipid mobilities in phosphatidylcholine-cholesterol membranes

Bimolecular collision rate of 8-anilinonaphthalene-1-sulfonic acid (ANS) and the nitroxide doxyl group attached to various carbons on stearic acid spin labels (n-SASL) in phosphatidylcholine-cholesterol membranes in the fluid phase was studied by observing dynamic quenching of ANS fluorescence by n-SASL's. The excited-state lifetime of ANS and its reduction by the n-SASL doxyl group were directly measured by the time-correlated single photon counting technique to observe only dynamic quenching separately from static quenching and were analyzed by using Stern-Volmer relations. The collision rate of ANS with the n-SASL doxyl group ranges between 1 X 10(7) and 6 X 10(7), and the extent of dynamic quenching by n-SASL is in the order of 5-much much greater than 6- greater than 7- less than 9- less than 10- less than 12- less than 16-SASL (less than 5-SASL) in dimyristoylphosphatidylcholine (DMPC) membranes. Collision rate of 16-SASL is only 10% less than that of 5-SASL. Since the naphthalene ring of ANS is located in the near-surface region of the membrane, these results indicate that the methyl terminal of SASL appears in the near surface area frequently, probably due to extensive gauche-trans isomerism of the methylene chain. The presence of 30 mol% cholesterol decreases the collision rate of ANS with 12- and 16-SASL doxyl groups but not with the 5-SASL doxyl group in DMPC membranes. On the other hand, in egg-yolk phosphatidylcholine membranes, inclusion of 30 mol% cholesterol does not affect the collision of ANS with either 5-SASL or 16-SASL doxyl groups, in agreement with our previous observation that alkyl chain unsaturation moderates cholesterol effects on lipid motion in the membrane (Kusumi et al., Biochim. Biophys. Acta 854, 307-317). It is suggested that dynamic quenching of ANS fluorescence by lipid-type spin labels is a useful new monitor of membrane fluidity that reports on various lipid mobilities in the membrane; a class of motion can be preferentially observed over others by selecting a proper spin label, i.e., rotational diffusion of lipid about its long axis and translational diffusion by using 5-SASL, wobbling motion of the lipid long axis by using 7-SASL or androstane spin label, and gauche-trans isomerism by using 16-SASL.