Objective To study the effect of fleroxacin （FLRX） on biological properties of Bloom （BLM） helicase catalytic core （BLM 642-2290 helicase） in vitro and the molecular mechanism of interaction between the two mol...Objective To study the effect of fleroxacin （FLRX） on biological properties of Bloom （BLM） helicase catalytic core （BLM 642-2290 helicase） in vitro and the molecular mechanism of interaction between the two molecules. Methods DNA-binding and unwinding activities of BLM 642-1290 helicase were assayed by fluorescence polarization and gel retardation assay under conditions that the helicase was subjected to different concentrations of FLRX. Effect of FLRX on helicase ATPase activity was analyzed by phosphorus-free assay based on a colorimetric estimation of ATP hydrolysis-produced inorganic phosphate. Molecular mechanism of interaction between the two molecules was assayed by ultraviolet and fluorescence spectra. Results The DNA unwinding and ATPase activities of BLM 642-1290 helicase were inhibited whereas the DNA-binding activity was promoted in vitro. A BLM-FLRX complex was formed through one binding site, electrostatic and hydrophobic interaction force. Moreover, the intrinsic fluorescence of the helicase was quenched by FLRX as a result of non-radioactive energy transfer. The biological activity of helicase was affected by FLRX, which may be through an allosteric mechanism and stabilization of enzyme conformation in low helicase activity state, disruption of the coupling of ATP hydrolysis to unwinding, and blocking helicase translocation on DNA strands. Conclusion FLRX may affect the biological activities and conformation of BLM 642-1290 helicase, and DNA helicase may be used as a promising drug target for some diseases.展开更多
The sensitized fluorescence intensity of terbium ion can be notably enhanced when the Tb 3+ fleroxacin complex is exposed to 365 nm light. By the measurements of fluorescence spectra, phosphorescence spectra, fl...The sensitized fluorescence intensity of terbium ion can be notably enhanced when the Tb 3+ fleroxacin complex is exposed to 365 nm light. By the measurements of fluorescence spectra, phosphorescence spectra, fluorescence quantum yield and fluorescence lifetime of the system, it is proved that irradiation makes the complex undergo a photochemical reaction and produces a new terbium complex which is more favorable to intramolecular energy transfer. The mechanism of the photochemical fluorescence enhancement was discussed.展开更多
基金supported by the National Basic Research Program of China (No.2010CB534912)the Doctoral Program of the Ministry of Education (No.200806570003)+1 种基金the Governor of Guizhou Province Talents Fund (No.200822)Guizhou University innovation fundsfor graduate student (No. Nongke 2012027)
文摘Objective To study the effect of fleroxacin （FLRX） on biological properties of Bloom （BLM） helicase catalytic core （BLM 642-2290 helicase） in vitro and the molecular mechanism of interaction between the two molecules. Methods DNA-binding and unwinding activities of BLM 642-1290 helicase were assayed by fluorescence polarization and gel retardation assay under conditions that the helicase was subjected to different concentrations of FLRX. Effect of FLRX on helicase ATPase activity was analyzed by phosphorus-free assay based on a colorimetric estimation of ATP hydrolysis-produced inorganic phosphate. Molecular mechanism of interaction between the two molecules was assayed by ultraviolet and fluorescence spectra. Results The DNA unwinding and ATPase activities of BLM 642-1290 helicase were inhibited whereas the DNA-binding activity was promoted in vitro. A BLM-FLRX complex was formed through one binding site, electrostatic and hydrophobic interaction force. Moreover, the intrinsic fluorescence of the helicase was quenched by FLRX as a result of non-radioactive energy transfer. The biological activity of helicase was affected by FLRX, which may be through an allosteric mechanism and stabilization of enzyme conformation in low helicase activity state, disruption of the coupling of ATP hydrolysis to unwinding, and blocking helicase translocation on DNA strands. Conclusion FLRX may affect the biological activities and conformation of BLM 642-1290 helicase, and DNA helicase may be used as a promising drug target for some diseases.
文摘The sensitized fluorescence intensity of terbium ion can be notably enhanced when the Tb 3+ fleroxacin complex is exposed to 365 nm light. By the measurements of fluorescence spectra, phosphorescence spectra, fluorescence quantum yield and fluorescence lifetime of the system, it is proved that irradiation makes the complex undergo a photochemical reaction and produces a new terbium complex which is more favorable to intramolecular energy transfer. The mechanism of the photochemical fluorescence enhancement was discussed.
