Patman is a fluorescent membrane probe related to Laurdan. The structural distinctions between the two probes are the lengths of the aliphatic tails (eleven carbons in Laurdan and fifteen in Patman) and the presence of a trimethylammonium group on Patman that produces a positively-charged head. Preliminary studies exploring Patman as a probe to detect membrane properties during apoptosis revealed that the fluorescence intensity of two edges of the emission spectrum (435 and 500 nm) stabilizes at different rates as the probe binds to the cell membrane. To test whether these differences represent dissimilarities in probe binding to ordered and disordered domains, experiments were conducted to monitor Patman equilibration with bilayers composed of various mixtures of saturated and unsaturated phosphatidylcholines at temperatures above, at, and below the main thermotropic phase transition. In general, Patman equilibrated more rapidly with bilayers in the liquid-disordered phase than in the solid-ordered phase. With solid phase membranes, the fluorescence stabilized faster at 500 nm than at 435 nm. Similar, yet more subtle, results occurred in the lipid disordered phase. In contrast, the situation was reversed at the phase transition temperature; equilibration was faster at 435 nm than at 500 nm. To determine whether these results reflected specific properties of Patman, the experiments were repeated with Laurdan, and several distinctions were observed. First, equilibration with solid phase lipids was faster than for Patman and not different from equilibration with the fluid phase. Second, differences in rates between the two wavelengths were less than with Patman for solid phase membranes but greater than with Patman for melted bilayers. Third, at the phase transition temperature, the difference in equilibration rates was the opposite of the result obtained with Patman. Computer simulations were used to assist with interpretation of these results. The data suggest that both probes bind superficially to the membrane before incorporating among the lipid molecules. Once within the membrane, Patman localizes to at least two distinct depths within the bilayer. Probe molecules in the shallow, more hydrated position favor 500 nm emission and those occupying a deeper, dehydrated position emit primarily at 435 nm. Laurdan's equilibration additionally represents movement of the probe between leaflets and multiple bilayers.
| Identifer | oai:union.ndltd.org:BGMYU2/oai:scholarsarchive.byu.edu:etd-4013 |
| Date | 12 May 2011 |
| Creators | Franchino, Hannabeth A. |
| Publisher | BYU ScholarsArchive |
| Source Sets | Brigham Young University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | http://lib.byu.edu/about/copyright/ |
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