Термолинзовая спектрометрия в исследовании гемопротеиновстатья

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[1] Термолинзовая спектрометрия в исследовании гемопротеинов / Д. С. Волков, К. И. Тищенко, Д. А. Недосекин и др. // Технологии живых систем. — 2010. — Т. 7, № 4. — С. 35–43. Сопоставлены оптические и термолинзовые спектры поглощения некоторых гемопротеинов (формы и комплексы гемоглобина и цитохрома c). Показано, что термолинзовая спектрометрия предоставляет широкие возможности как для определения характеристик полос поглощения белков, так и обнаружения фотохимических реакций, индуцированных лазерным излучением, при этом высокая точность измерений спектров поглощения наблюдается как в растворах, так и в составе клеточных структур. Thermal-lens spectrometry (TLS) is one of the most sensitive methods of molecular-absorption spectroscopy complementing conventional methods, as spectrophotometry and IR spectroscopy measure the transmittance of the electromagnetic radiation, while TLS is based on nonradiative transfers of excited molecules caused by the absorbed part of the radiation going through a sample. Thermal relaxation of the absorbed energy results in sample heating and the formation of a nonuniform spatial profile of the refractive index (a thermal lens). The optical strength of the thermal lens is directly proportional to the absorbance of the sample and, thus, to the concentration and absorption coefficient of the studied compound. By increasing the power of the excitation beam, it is possible to achieve the same high sensitivity as in luminescence analysis but for nonfluorescent molecules. Methods of TLS provide the detection of absorbances in liquids of 10-9 - 10-6 abs. units (corresponding concentrations of 10-11 mol L-1) and to analyze volumes of 10-12 L with as low as several absorbing molecules in this volume. If processes of the dissipation of the absorbed radiation other than thermal relaxation exist (luminescence or photochemical processes), the strength of the thermal-lens effect is decreased as expected, and this phenomenon can be used for elucidating such processes and estimating its quantum yields. The aim of this paper was (1) to estimate the possibilities of thermal-lens spectrometry for relevant determination of the absorption-band parameters of heme proteins (forms and complexes of haemoglobin and cytochrome c) and (2) by the examples of photochemically stable and unstable protein species, to show that a comparison of optical and thermal-lens measurements can be used for revealing photochemical processes in the system in question, which is important for studying laser and diode radiation effect on biological materials. We compare optical and thermal-lens absorptions spectra of some heme proteins (forms and complexes of haemoglobin and cytochrome c). It is shown that thermal-lens spectrometry shows wide potentialities both for the determining the absorption-band parameters of proteins and for detecting laser-induced photochemical reactions; high precision of the measurements of absorption spectra is observed both in solutions and in cellular structures. For haemoglobin and cytochrome c we estimated the amounts of proteins providing the determination of molar absorptivities from thermal-lens measurements with enough precision. For all the species studied it was 0.1-0.2 pmol (1-2 ng) of proteins. For a cardiolipin-cytochrome c-NO complex, for which previous studies relaibly show mainly photochemical rather than He thermal effect of the laser radiation, we estimated the quantum yield of the photolysis reaction from thermal-lens measurements, 0.45 ± 0.08.

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