Laurdan Spectrum Decomposition as a Tool for the Analysis of Surface Bilayer Structure and Polarity: a Study with DMPG,Peptides and Cholesterol |
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Authors: | Aline D Lúcio Cíntia C Vequi-Suplicy Roberto M Fernandez and M Teresa Lamy |
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Institution: | 1.Instituto de Física,Universidade de S?o Paulo,S?o Paulo,Brazil;2.Departamento de Exatas,Universidade Federal de Lavras,Lavras,Brazil;3.Depto. de Física e Biofísica,Instituto de Biociências da Universidade Estadual Paulista,Botucatu,Brazil |
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Abstract: | The highly hydrophobic fluorophore Laurdan (6-dodecanoyl-2-(dimethylaminonaphthalene)) has been widely used as a fluorescent
probe to monitor lipid membranes. Actually, it monitors the structure and polarity of the bilayer surface, where its fluorescent
moiety is supposed to reside. The present paper discusses the high sensitivity of Laurdan fluorescence through the decomposition
of its emission spectrum into two Gaussian bands, which correspond to emissions from two different excited states, one more
solvent relaxed than the other. It will be shown that the analysis of the area fraction of each band is more sensitive to
bilayer structural changes than the largely used parameter called Generalized Polarization, possibly because the latter does
not completely separate the fluorescence emission from the two different excited states of Laurdan. Moreover, it will be shown
that this decomposition should be done with the spectrum as a function of energy, and not wavelength. Due to the presence
of the two emission bands in Laurdan spectrum, fluorescence anisotropy should be measured around 480 nm, to be able to monitor
the fluorescence emission from one excited state only, the solvent relaxed state. Laurdan will be used to monitor the complex
structure of the anionic phospholipid DMPG (dimyristoyl phosphatidylglycerol) at different ionic strengths, and the alterations
caused on gel and fluid membranes due to the interaction of cationic peptides and cholesterol. Analyzing both the emission
spectrum decomposition and anisotropy it was possible to distinguish between effects on the packing and on the hydration of
the lipid membrane surface. It could be clearly detected that a more potent analog of the melanotropic hormone α-MSH (Ac-Ser1-Tyr2-Ser3-Met4-Glu5-His6-Phe7-Arg8-Trp9-Gly10-Lys11-Pro12-Val13-NH2) was more effective in rigidifying the bilayer surface of fluid membranes than the hormone, though the hormone significantly
decreases the bilayer surface hydration. |
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