Triplet-photochrome method

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 medium 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 labeled proteins and probes in a medium of high viscosity (such as biomembranes), a new triplet-photochrome labeling technique has been developed [5, 8, 12, 17]. 

The stilbene photoisomerization through the triplet potential surface can be sensitized by a donor molecule excited to its triplet state, which is energetically dose to the stilbene exdted triplet level Ti ([52, 53]. The sensitizers (donors) with triplet energies of at least 255kJ/mol (in a case of unsubstituted stilbene) transfer their energies to both trans and cis isomers of the stilbene molecule in the ground state in a diffusion-controlled process. The reaction proceeds from an initial 

The triplet-photochrome method is based on the above-mentioned scheme. Starting from cis-stilbene, which is not fluorescent at the steady-state conditions, and measuring the rate of increase of emitted fluorescence, it has been possible to monitor the process of the sensitized cis-trans photoisomerization. The cis-stilbene concentration, which is proportional to fluorescence intensity, approaches the photo stationary level exponentially with the rate constant 

Due to the relatively long lifetime of the sensitizer triplet state and the possibility of integrating data on the stilbene photoisomerization, the apparent characteristic time of the method can reach hundreds of seconds. This unique property of the triplet-photochrome technique allows the investigation of slow diffusion processes, including encounters of proteins in membranes using very low concentrations of both the triplet and photochrome probes. 

The triplet-photochrome labeling method has been used to study very rare encounters in a system containing the erythrosin B sensitizer and SITC photochrome probe [12]. Both types of molecules were covalently bound to a-chymotryp-sin. The photoisomerization kinetics was monitored by fluorescence decay of the frans-SITS. The rate constants of the triplet-triplet energy transfer between eryth-rosinBandSITS (at room temperature and pH 7) were found to be k J = 10 M s . It should be emphasized that the concentration of the triplet sensitizer attached to the protein did not exceed 10 M in those experiments, and the collision frequencies were close to 10 s that are eight to nine orders of magnitude less than those measured with the regular fluorescence or ESR techniques. 

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The triplet-photochrome method is based on the above mentioned cascade scheme. Starting from ctT-stilbene, which is not fluorescent at the steady-state conditions of our... 

Papper, V., Medvedeva, N., Fishov, I., and Likhtenshtein, G.I. (2000) Quenching of cascade reaction between triplet and photochrome probes with nitroxide radicals novel labeling method in study ofmembranes and surface system, 7. Appl. Biotech. Biophys. 88, 1-17. 

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