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RISS 인기검색어
- 검색키워드
- 저자 : Klymchenko, Andrey S. (검색결과 2 건)
- 무료
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Boudier, Christian,Klymchenko, Andrey S.,Mely, Yves,Follenius-Wund, Anny Korean Society of Photoscience 2009 Photochemical & photobiological sciences Vol.8 No.6
The complex multistep inhibition of proteinases by $alpha_1$-antitrypsin (${\alpha}_1$-AT) was investigated by covalently labeling its unique Cys residue with a ratiometric environment-sensitive fluorescent dye, 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone (BMFC). The binding of BMFC-labeled ${\alpha}_1$-AT with pancreatic elastase led to significant changes in the dual emission of BMFC. The 8 nm blue shift of one of the bands and ca. 65% change in the intensity ratio of the two emission bands suggested an increased exposure of the labeled Cys-232 residue to the bulk water on complex formation. In contrast, the bacterial V8 proteinase-induced cleavage of the reactive center loop of BMFC-labeled ${\alpha}_1$-AT did not generate any significant change in the Cys-232 region. Similar experiments with elastase and ${\alpha}_1$-AT conjugated to the classical environment-sensitive dye, IANBD, confirmed these results but led to much smaller modifications in the emission spectrum. Stopped-flow investigation of the reaction between BMFC-labeled ${\alpha}_1$-AT and elastase showed both a well-described fast and a new slow step of the inhibition process. The latter step is probably associated with the structural reorganization aimed at stabilizing the final complex. These results present a convenient fluorescence ratiometric approach based on the BMFC label for studies of protein conformational changes.
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Unusually slow proton transfer dynamics of a 3-hydroxychromone dye in protic solvents
Das, Ranjan,Klymchenko, Andrey S.,Duportail, Guy,Mely, Yves Korean Society of Photoscience 2009 Photochemical & photobiological sciences Vol.8 No.11
The photophysics of a 3-hydroxychromone dye, 2-(2-furyl)-3-hydroxychromone (FHC) was explored in different types of protic solvents by steady-state and time-resolved fluorescence spectroscopy. FHC exhibits a dual emission, attributable to the excited normal ($N^*$) and tautomer ($T^*$) forms resulting from an excited state intramolecular proton transfer (ESIPT) reaction ($N^*{\rightarrow}T^*$). The ESIPT rate decreases with an increase in the hydrogen-bond donating ability of protic solvents. The proton-transfer dynamics is found to be unusually slow (${\sim}10^3$ times slower than 3-hydroxyflavone) in strong hydrogen-bond donating solvents like methanol, trifluoroethanol or formamide, where it occurs on a time scale of ~100-260 picoseconds. This slow dynamics is likely to be related to the intermolecular solvent.solute H-bonding interactions that compete with the intramolecular H-bond in FHC required for the ESIPT reaction. These data provide a physical background for recent applications of this dye as a fluorescence probe of the microenvironment in biomolecules.
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