Mobility of fluorescent probe molecules in lipid bilayer vesicles as studied by steady-state and time-dependent nuclear Overhauser effect measurements in 1H-nuclear magnetic resonance spectroscopy

Abstract

In order to elucidate the mobilities of the fluorophores of fluorescent 2- and 16-(9-anthroyloxy)palmitic acids (16-AP and 2-AP, respectively) in lipid bilayer vesicles, the steady-state and time-dependent nuclear Overhauser effects in 1H-NMR spectroscopy, but not the fluorescence depolarization in fluorescence spectroscopy, have been measured. The steady-state nuclear Overhauser effect measurements showed an appreciable magnitude of negative nuclear Overhauser effects between the resonances due to the fluorophores of the two fluorescent probes and lipids. These results definitely mean that in lipid bilayers, the fluorophores (anthroyloxy ring) of the fluorescent probes experience other types of motions with much longer correlation times than those detected by the fluorescence depolarization measurements, since at the correlation time showed by the fluorescent method (1–2 · 10 −9 s or less), no such transfer of the negative nuclear Overhauser effects is expected to occur. The correlation times of the fluorophores, as calculated from the cross-relaxation rates of the anthroyl ring protons of 16-AP and 2-AP, were 3.8 · 10 −8 and 1.1 · 10 −7 s, respectively. These values, respectively, compare favorably with those of the terminal methyl of acyl chains and the choline methyl carbons which were estimated by 13C T 2 relaxation times. Thus, it is concluded that the fluorophores of both 16-AP and 2-AP have a slow form of motion which moves with a similar time scale to those of lipids in addition to the faster one that causes fluorescence depolarization.