Wavelength dependence of patman equilibration dynamics in phosphatidylcholine bilayers

Abstract

Assessment of the equilibration kinetics of Patman at the edges of its emission spectra provided additional insights about membrane properties beyond those obtained from end-point fluorescence measurements. Upon introduction of the probe to aqueous suspensions of liposomes, the emission intensity slowly increased about 10-fold (t(½)=~100 s). The rate of equilibration depended on emission wavelength, and was usually faster at 500 than at 435 nm. However, this trend was reversed for equilibration with lipids at their phase transition temperature. The apparent rotational motion of the dye also differed between the long and short emission wavelengths but did not display the slow equilibration time dependence observed with intensity measurements. These results suggested that slow equilibration reflects relaxation of the immediate membrane microenvironment around the probe rather than slow insertion into the membrane. The data were rationalized with a model that allows two membrane/probe configurations with distinct microenvironments. The analysis suggests that by monitoring the equilibration pattern of Patman, inferences can be made regarding the polarity of two microenvironments occupied by the probe, the distribution of the probe among those microenvironments, and the kinetics with which they relax to equilibrium.

Relationships between membrane water molecules and Patman equilibration kinetics at temperatures far above the phosphatidylcholine melting point

“…All of the experimental methods used in this study have been described in [20]. Lipid stocks (all purchased from Avanti Polar Lipids, Alabaster, AL) were dissolved in chloroform then dried under a nitrogen stream followed by high vacuum.…”

“…For simplicity, it is assumed that when the probe is mixed with an aqueous suspension of phospholipid vesicles, all of the probe molecules immediately adsorb to the membrane surface [20]. Regardless of whether this assumption is valid, we have avoided the issue experimentally by not varying the concentrations of probe and membrane lipid.…”

“…For these measurements, we turned our attention to Laurdan since it would be sensitive to changes in membrane order [17]. Patman was not used for these studies because the anchoring effects of the trimethylammonium group and the longer acyl chain blunt the sensitivity of anisotropy measurements to membrane dynamics [20]. As shown in Fig.…”

“…We have considered the hypothesis proposed above (faster water rotation) as well as an alternative that these effects result instead from increases in the number and/or penetration depth of bilayer water molecules. We take advantage of recent observations from kinetic analyses of probe equilibration suggesting that these naphthalene derivatives may reside in at least two configurations in the membrane and that one can learn additional detail about membrane dynamics by considering the properties of those configurations [20,21]. For such studies, Patman appears ideally suited [20], and we have therefore focused our attention on the behavior of that probe with unilamellar vesicles of various saturated and unsaturated phosphatidylcholines.…”

“…We take advantage of recent observations from kinetic analyses of probe equilibration suggesting that these naphthalene derivatives may reside in at least two configurations in the membrane and that one can learn additional detail about membrane dynamics by considering the properties of those configurations [20,21]. For such studies, Patman appears ideally suited [20], and we have therefore focused our attention on the behavior of that probe with unilamellar vesicles of various saturated and unsaturated phosphatidylcholines. The results unexpectedly provided evidence that phospholipid behavior is more diverse at temperatures far about the phase transition than previously reported.…”