Two-Photon Fluorescence Microscopy Imaging of Cellular Oxidative Stress Using Profluorescent Nitroxides

A range of varying chromophore nitroxide free radicals and their nonradical methoxyamine analogues were synthesized and their linear photophysical properties examined. The presence of the proximate free radical masks the chromophore’s usual fluorescence emission, and these species are described as profluorescent. Two nitroxides incorporating anthracene and fluorescein chromophores (compounds 7 and 19, respectively) exhibited two-photon absorption (2PA) cross sections of approximately 400 G.M. when excited at wavelengths greater than 800 nm. Both of these profluorescent nitroxides demonstrated low cytotoxicity toward Chinese hamster ovary (CHO) cells. Imaging colocalization experiments with the commercially available CellROX Deep Red oxidative stress monitor demonstrated good cellular uptake of the nitroxide probes. Sensitivity of the nitroxide probes to H2O2-induced damage was also demonstrated by both one- and two-photon fluorescence microscopy. These profluorescent nitroxide probes are potentially powerful tools for imaging oxidative stress in biological systems, and they essentially “light up” in the presence of certain species generated from oxidative stress. The high ratio of the fluorescence quantum yield between the profluorescent nitroxide species and their nonradical adducts provides the sensitivity required for measuring a range of cellular redox environments. Furthermore, their reasonable 2PA cross sections provide for the option of using two-photon fluorescence microscopy, which circumvents commonly encountered disadvantages associated with one-photon imaging such as photobleaching and poor tissue penetration

APPLICATION OF THE MÖSSBAUER EFFECT TO THE INVESTIGATION OF DYNAMIC STRUCTURE AND CATALYTIC PROPERTIES OF ENZYMES

La mobilité locale de protéines a été étudié à l'aide des marqueurs Mössbauer, spin ou proton. L'augmentation de la tempéature des solutions de protéines et l'accroissement du degré d'hydratation d'échantillons anhydres diminuent le facteur f ' des noyaux Mössbauer situés aux centres actifs ou liés à la matrice de protéine (α-chymotrypsine, séroalbumine). La diminution de f ' est due apparemment à des mouvements spontanés de coupure des chaînes polypeptidiques par suite des fluctuations thermiques. On a trouvé une corrélation entre les propriétés dynamiques, le degré d'hydratation et l'activité catalytique de l'α-chymotrypsine. Le temps de corrélation des mouvements de coupure dans les protéines est de l'ordre de 10-11 à 10-7 s ainsi qu'on le déduit de la mobilité des marqueurs par RGN, RPE et RMP. Les modifications spontanées de conformation peuvent être des mécanismes d'adaptation dynamique et de transitions allostériques et transglobulaires.The local mobility of the protein macromolecules has been studied by using the set of the Mössbauer, spin and proton labels. The increase in temperature of the protein solutions and the increase in hydration degree of dry samples result in the decrease in f ' of the Mössbauer nuclei, entering the active centres or bounding with the protein matrix (α-chymotrypsin, serum albumin). The decrease in f ' is accounted apparently by spontaneous segmental motions of polypeptide chains as result of heat fluctuations. The correlation between the dynamic properties, hydration degree and catalytic activity of α-chymotrypsin was found. The correlation time of segmental motions in the proteins is found in the region 10-11-10-7 s by analysis of mobility of the labels by means of gammaresonance spectroscopy, ESR and PMR. The spontaneous conformational changes may provide mechanisms of the dynamic adaptation, allosteric and transglobular transitions