SPECTRAL PROPERTIES OF FLUORESCEIN IN SOLVENT-WATER MIXTURES - APPLICATIONS AS A PROBE OF HYDROGEN-BONDING ENVIRONMENTS IN BIOLOGICAL-SYSTEMS

Although fluorescein is a widely used fluorescent probe in the bioscie nces, the effect of solvent environment on its spectral properties is poorly understood. In this paper we explore the use of fluorescein as a probe of the state of hydrogen bonding in its local environment. Thi s application is based on the observation, originally made by Martin ( Chem. Phys. Lett. 35, 105-111, 1975), that the absorption maximum of f luorescein undergoes substantial shifts in organic solvents related to the hydrogen bonding power of the solvents. We have extended this wor k by studying the spectral properties of the dianion form of the probe in solvent-water mixtures. We show that the magnitude of the shift co rrelates with the alpha and beta parameters of Kamlet and Taft (J. Am. Chem. Soc. 98, 377-383; 2886-2894, 1976), which provide a scale of th e hydrogen bond donor acidities and acceptor basicities, respectively, of the solvents. In solvent-water mixtures, these shifts reflect gene ral effects of the solvents on the hydrogen bonding environment of the fluorescein through water-solvent hydrogen bonding and specific effec ts due to fluorescein-solvent hydrogen bonding. Indeed, both the absor ption and fluorescence properties appear to be dominated by these effe cts indicating that the spectral shifts of the dianion can be used as an indicator of its hydrogen bonding environment. We discuss the appli cation of fluorescein as a probe of hydrogen bonding in the microenvir onment immediately surrounding the fluorophore, and we illustrate the effect with reference to the fluorescein-antifluorescein antibody comp lex where it appears that antibodies selected during the immune respon se possess binding sites that are increasingly dehydrated and hydropho bic.