Spin-Triplet-Photochrome Methods

Spin probe Triplet probe Photochrome probe 

The efTective rate constant of the triplet-triplet energy transfer (k) in membrane can be calculated from the relation between apparent (experimental) rate constants kapp for probes in solution and in a membrane 

Experimental results gave the evidence of an efficient nitroxide radical inhibitor effect upon the sensitized cis-trans photoisomerization of DMAAS by quenching the sensitizer triplet state. The phosphorescence triplet lifetime of erythrosin B in PPDC membranes was measured to be 3 x 10 s. The experimental quenching rate constant of the cascade reaction kq and the rate constant of the triplet-triplet energy transfer kx evaluated in 2D terms were obtained as kq = (1.05 0.08) X 10 cm /(mols) and kx = (1.26 0.21) x 10 cm /(mols). Taking into consideration the efficiency of the triplet-triplet energy transfer y = 3.5 %, the rate constant of encounters between the sensitizer and the photochrome was found as ke = 3.4x 10 cm /(mols). 

The expanded cascade method includes quenching of the excited triplet state of (Er) hy nitroxides at local concentrations starting from 1 pM. Such a combination maintains some facilities of the above-mentioned probes and has an essential advantage in the study of molecular dynamics and measurements of the local concentration of radicals. Due to the relatively long lifetime of the sensitizer triplet state, which represents the timescale of the method, and due to the opportunity to integrate the data on stilbene isomerization, the apparent characteristic time of the cascade method may reach hundreds of seconds. This property of the cascade system allows investigation of low-diffusion processes using very low concentration of probes. 

In the frame of CSTPM, the following dynamics parameters of the cascade system components can be experimentally measured the spin label rotation correlation time and spin relaxation parameters, the fluorescence and phosphorescence polarization correlation times, the singlet and triplet state quenching rate constants, the rate constant of photoisomerization, and the rate constant of the triplet-triplet energy transfer. This set of parameters is a cumulative characteristic of the dynamic state of biomembranes in the wide range of the probes amplitude and characteristic time. 

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The traditional fluorescence and electron-spin resonance methods for recording molecular collisions do not allow the study of translational diffusion and rare encounters of molecules in a viscous media because of the short characteristic times of these methods. To measure the rate constants of rare encounters between macromolecules and to investigate the translation diffusion of labelled proteins and probes in a medium of high viscosity (like biomembranes), a new triplet-photochrome labeling technique has been developed (Mekler and Likhtenshtein, 1986 Mekler and Umarova, 1988 Likhtenshtein, 1993 Papper and Likhtenshtein, 2001). 

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