Quenching of Cascade Reaction Between Triplet and Photochrome Probes with Nitroxide Radicals

Abstract

We proposed a new method for the study of molecular dynamics and fluidity of the living and model biomembranes and surface systems. The method is based on the measurements of the sensitized photoisomerization kinetics of a photochrome probe. The cascade triplet cis-trans photoisomerization of the excited stilbene derivative sensitized with the excited triplet Erythrosin B has been studied in a model liposome membrane. The photoisomerization reaction is depressed with nitroxide radicals quenching the excited triplet state of the sensitizer. The enhanced fluorescence polarization of the stilbene probe incorporated into liposome membranes indicates that the stilbene molecules are squeezed in a relatively viscous media of the phospholipids. Calibration of the "triple" cascade system is based on a previously proposed method that allows the measurement of the product of the quenching rate constant and the sensitizer's triplet lifetime, as well as the quantitative detection of the nitroxide radicals in the vicinity of the membrane surface. The experiment was conducted using the constant-illumination fluorescence technique. Sensitivity of the method using a standard commercial spectrofluorimeter is about 10(-12) mol of fluorescence molecules per sample and can be improved using an advanced fluorescence technique. The minimal local concentration of nitroxide radicals or any other quenchers being detected is about 10(-5) M. This method enables the investigation of any chemical and biological surface processes of microscopic scale when the minimal volume is about 10(-3) microL or less.

Novel Fluorescent Methods for Biotechnological and Biomedical Sensoring: Assessing Antioxidants, Reactive Radicals, NO Dynamics, Immunoassay, and Biomembranes Fluidity

“…A cascade of photochemical and photophysical reactions between a triplet sensitizer and a fluorescence-photochrome probe exhibiting the phenomena of cis-trans photoisomerization, triplet-triplet energy transfer, and the triplet exited-state quenching by a stable radical has been proposed and developed [2,[32][33][34][35][36].…”

“…Thus, by measuring the kinetic photoisomerization of the stilbene derivative molecules and proper calibration, one can acquire information about the microviscosity and steric hindrance of their surrounding environment. This method was used for the measurement fluidity of biological membranes [2,[31][32][33][34]. Information about the microviscosity of the binding site region is obtained when the label is bound to a specific site of a protein (as for an antigen, in a binding site of an antibody, or for a substrate, in the binding site of an enzyme, etc.)…”

“…The method was employed for studying molecular dynamics of biological membranes [2,[31][32][33][34][35]. It was shown that PSS exhibits fluorescent characteristics that are similar to those of typical membrane fluorescence probes such as diphenylhexatriene and, therefore, is suitable for studying molecular dynamics and micropolarity of media using the steady-state and time-domain fluorescent polarization and the spectral relaxation shift techniques.…”

Photophysical Properties of the Prefluorescent Nitroxide Probes QT and C343T

“…The radical trapping by the nitroxide moiety leads to the diamagnetic alkoxyamine, thereby eliminating the intramolecular quenching and restoring the fluorescence of the chromophore (9,10). These probes act as molecular switches allowing a direct quantification of free radical formation at concentrations lower than the detection limit of standard electron spin resonance techniques (ranging from 10 )4 to 10 )7 M M) (3,7,11). Thus, nitroxide prefluorescent probes have been employed to measure radical concentrations generated by pulse laser photolysis or to monitor radical processes in thin polymer films and zeolites (3,(6)(7)(8).…”

A Fluorescent-Photochrome Method for the Quantitative Characterization of Solid Phase Antibody Orientation

“…Moreover, they possess absorption and fluorescence properties similar to typical biochemical probes that can be monitored by simple optics. In addition, the quantitative study of direct and sensitized photoisomerization of stilbenes opens up new possibilities for the measurement of rotational and translational diffusion, both of which depend on molecular dynamics and steric hindrance in the vicinity of the probe (17)(18)(19).…”