Experimental and theoretical studies of the electronic spectra of Prodan and Laurdan in solvents and lipid bilayers

Author(s)

Cíntia Cristina de Vequi Suplicy

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Física (IF/SBI)
• Defense date: 2010-09-13

Examining board members

Maria Teresa Moura Lamy; Tertius Lima da Fonseca; Cássia Alessandra Marquezin; Elisabeth Andreoli de Oliveira; Hubert Karl Stassen

• Advisor: Maria Teresa Moura Lamy; Kaline Rabelo Coutinho

Abstract

Prodan (6-propionyl-2-dimethylamino-naphthalene) and Laurdan (6-dodecanoyl-2- dimethylamino-naphthalene) molecules are frequently used as fluorescent probes in lipid bilayers and, more rarely, in cell membranes. The objective of this PhD thesis was to study these probes in several environments, to increase the understanding about its structures and spectroscopic characteristics in several solvents and lipid bilayers. This study was carried out using two approaches: experimental and theoretical. With experimental and theoretical results, we demonstrated that the two molecules have the same chromophore. Experimentally, electronic absorption spectra were measured and compared in solvents of several polarities and lipid bilayers, for both molecules. A theoretical model was constructed for Prodans fundamental state in several solvents, in which we found that its molecular geometry is planar and that the molecule undergoes a great deal of polarization in solvents of higher polarity. Theoretically, we emulated the absorption spectra in all solvents studied, showing that they are well described by three electronic transitions. We verified, experimental and theoretically, that Prodan aggregates in aqueous solution at all concentration ranges (0.9 a 20.0 uM). Additionally, the fluorescent emission spectra were measured and compared, and we demonstrated that they are composed by two emission bands in all solvents and lipid bilayers. These bands were related to two fluorescent lifetimes, evidencing the presence of two excitation states for these probes. However, the nature of the excited states in homogeneous solvents and in lipid bilayers seems to be different. A methodology to analyze the emission spectra was proposed. The comparison of Prodans and Laurdans emission spectra in lipid bilayers showed that the probes monitor a slightly different micro-environment. Our results indicated that both fluorophores are located near the bilayers surface, although Prodan seems to be more exposed to water molecules. Besides that, Prodan partitions in water, when the bilayer is in the gel phase. (AU)