LOCATION OF DIPHENYLHEXATRIENE (DPH) AND ITS DERIVATIVES WITHIN MEMBRANES - COMPARISON OF DIFFERENT FLUORESCENCE QUENCHING ANALYSES OF MEMBRANE DEPTH

The average membrane location of a series of diphenylhexatriene (DPH)- derived membrane probes was analyzed by measuring the quenching of DPH fluorescence with a series of nitroxide-labeled lipids in which the d epth of the nitroxide group is varied. All DPH derivatives were locate d deeply within the bilayer. Some derivatives were anchored at a shall ower depth than free DPH by attachment to cationic or anionic groups. However, the absolute change in DPH depth upon attachment to such grou ps was relatively modest (<4 Angstrom). In fact, protonated DPH fatty acid and a DPH fatty acyl group attached to a phosphatidylcholine were found to locate slightly more deeply than free DPH. The location of D PH derivatives can be explained by the length of the DPH group and its tendency to orient predominantly parallel to the fatty acyl chains of the bilayer. These factors allow a charged group attached to one end of a DPH molecule to be accommodated at the polar surface while mainta ining a deep DPH location. Basically, it appears that most DPH derivat ives probe the same region in the bilayer. We conclude previously repo rted differences in fluorescence polarization of free and anchored for ms of DPH may reflect a direct effect of anchoring on motion rather th an an effect on average DPH location. Other experiments showed the loc alization of DPH probes was found to be similar in the presence and ab sence of cholesterol. This implies that previously observed cholestero l-induced effects on DPH fluorescence polarization also largely reflec t differences in DPH motion, not DPH location. From the quenching resu lts it was also possible to define rules governing the location of a v ariety of chemical groups in membranes by comparison of the results ob tained with DPH derivatives to those of similar derivatives of other f luorescent groups. Finally, an important goal of this study was to com pare different methods of analysis of quenching data: parallax analysi s, distribution (Gaussian) analysis (using a single Gaussian), and a s econd-order polynomial analysis. To evaluate the accuracy of these met hods, the apparent depths of a series of fluorescence probes previousl y analyzed by parallax analysis was reanalyzed with all three methods. There was good agreement unless the fluorescent molecule was very sha llow or very deep. In such cases, only parallax analysis gave physical ly reasonable results. This is likely to be due to the lack of a suffi cient number of quenchers spanning a wide enough range for other analy ses to compensate for deviations from ideal curves. Parallax analysis was also compared to distribution (Gaussian) analysis using a double G aussian fit to account for quenching from the trans leaflet (Ladokhin, A. (1997) Methods Enzymol. 278, 462-473). Again more physically reaso nable results were obtained from parallax analysis, likely due to non- Gaussian behavior of the depth dependence of quenching. Notwithstandin g these observations, the significant number of cases where Gaussian c urve fitting methods for quenching analysis are most powerful are disc ussed.