The photophysics and photochemistry of pefloxacin(PEF),a 1-ethyl-substituted fluoroquinolone(FQ)antibiotic,were studied using transient,steady-state experimental methods and computational methods.The fundamental photo...The photophysics and photochemistry of pefloxacin(PEF),a 1-ethyl-substituted fluoroquinolone(FQ)antibiotic,were studied using transient,steady-state experimental methods and computational methods.The fundamental photoproperties of PEF and its phototoxicity toward lysozyme,a single-chain protein,were compared with those of a 1-fluorophenyl-substituted FQ antibiotic,difloxacin(DIF).The results showed that the phototoxicity was significantly decreased by the insertion of the bulky1-fluorophenyl substituent(the phototoxicity of DIF was approximately one-quarter of that observed for PEF).This trend was attributed to the lowest lying singlet state with sizeable oscillator strength(f C 0.1)being shifted from319 nm in PEF to 266 nm in DIF upon the insertion of the bulky substituent at the 1-position,as investigated by using computational methods.In addition,95%of the solar UV irradiation that reaches the earth’s surface has wavelength[315 nm.Therefore,reducing the most effective excitation wavelength by optimizing the substituent at the1-position may be a promising strategy to alleviate the phototoxicity of FQ antibiotics.These findings may be applied to other FQ antibiotics because a large number of phototoxicity studies on FQ antibiotics with different substituents at the 1-position can prove these finding’s effectiveness.Delafloxacin,an FQ antibiotic bearing a chlorine and bulky substituent at the 8-and 1-positions,respectively,exhibits no phototoxicity is the most recent example reported to date.To the best of our knowledge,this is the first transient and steady-state study of the effect of the N-1 substituent on the photochemistry and phototoxicity of FQ antibiotics.These findings will be beneficial to the development of novel FQ antibiotics without phototoxicity.展开更多
To identify the unknown proteins that would contaminate the α- and β-subunits of nitrogenase MoFe protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, the partially purified MoFe prote...To identify the unknown proteins that would contaminate the α- and β-subunits of nitrogenase MoFe protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, the partially purified MoFe protein （Avl） preparation was obtained from Azotobacter vinelandii Lipmann OP by chroma- tography on DEAE-cellulose （DE52） and Sephacryl S-200 columns and analyzed by PAGE and matrix- assisted laser desorption/ionization time-of-flight （MALDI-TOF） mass spectrometry. The Av 1 preparation was shown to have two main bands at the position of the α- and β-subunits of crystalline Avl on the SDS gel. However, on the anoxic native PAGE, in addition to the Avl band, the preparation was shown to have three other main bands that migrated slower than Av 1. Of these three main bands, the protein with the fastest migration was identified as bacterioferritin elsewhere. The proteins on the other two bands, termed Upper and Middle, were suggested to be two different homopolymers with the same apparent subunit electrophoretic mobilities as the α- and β-subunits of Avl, respectively. By analysis of MALDI-TOF mass spectrometry, the Upper was identified as GroEL, which belongs to the heat shock protein 60 family, and the Middle was identified as glucose-6-phosphate isomerase （PGI）. In our preparation, anoxic native electrophoresis indicated that GroEL was composed of 14 identical subunits and that PGI was composed of 10 identical subunits. This is the first report of PGI, with so many subunits. The contaminating proteins in the Av 1 preparation, mainly GroEL and PGI, could be totally or partially removed from Av 1 if the shoulders and center of the elution peak were collected separately from the Sephacryl S-200 column and the center fraction was purified further by Q-Sepharose developed with an NaC1 concentration gradient. Thus, Avl with more than 90% purity was obtained. Obviously, this modified method is useful for the purification of mutant MoFe proteins with a high purity.展开更多
A new protein, an approximately 59-kDa monomer containing iron atoms, was first isolated from the mutant strain DJ35 of Azotobacter vinelandii Llpmann. After analysis by matrix-assisted laser desorptlon ionization tim...A new protein, an approximately 59-kDa monomer containing iron atoms, was first isolated from the mutant strain DJ35 of Azotobacter vinelandii Llpmann. After analysis by matrix-assisted laser desorptlon ionization time-of-flight mass spectrometry, the protein was Identified as the product of a predicted gene. Thus, the protein was tentatively called HBP59. Its absorption spectra （ABS） In the reduced state exhibited three peaks at 421,517, and 556 nm and the maximal peak was shifted from 421 to 413 nm after exposure of HBP59 to air. The Soret circular dichrolsm （CD） spectrum of HBP59 In the reduced state displayed four positive peaks at 364, 382, 406, and 418 nm and two negative peaks at 398 and 433 nm; the △ε （CD extinction coefficient） values of these peaks were found to be 0.92, 0.58, 0.87, 0.72, -0.65 and -1.12 L/mol per cm, respectively. Titration with heme showed that the protein has 0.1 heme molecules/protein molecule. After HBP59 had fully Interacted with heme, Its maximal ABS value and Soret CD Intensity were increased by approximately 10-fold compared with values before Interaction. Therefore, It seems that one molecule of HBP59 can be interacted with only one heme. These results indicate that HBP59 contains heme with low spin and may be Involved In heme utilization or adhesion.展开更多
基金supported by the National Natural Science Foundation of China(No.21173252)。
文摘The photophysics and photochemistry of pefloxacin(PEF),a 1-ethyl-substituted fluoroquinolone(FQ)antibiotic,were studied using transient,steady-state experimental methods and computational methods.The fundamental photoproperties of PEF and its phototoxicity toward lysozyme,a single-chain protein,were compared with those of a 1-fluorophenyl-substituted FQ antibiotic,difloxacin(DIF).The results showed that the phototoxicity was significantly decreased by the insertion of the bulky1-fluorophenyl substituent(the phototoxicity of DIF was approximately one-quarter of that observed for PEF).This trend was attributed to the lowest lying singlet state with sizeable oscillator strength(f C 0.1)being shifted from319 nm in PEF to 266 nm in DIF upon the insertion of the bulky substituent at the 1-position,as investigated by using computational methods.In addition,95%of the solar UV irradiation that reaches the earth’s surface has wavelength[315 nm.Therefore,reducing the most effective excitation wavelength by optimizing the substituent at the1-position may be a promising strategy to alleviate the phototoxicity of FQ antibiotics.These findings may be applied to other FQ antibiotics because a large number of phototoxicity studies on FQ antibiotics with different substituents at the 1-position can prove these finding’s effectiveness.Delafloxacin,an FQ antibiotic bearing a chlorine and bulky substituent at the 8-and 1-positions,respectively,exhibits no phototoxicity is the most recent example reported to date.To the best of our knowledge,this is the first transient and steady-state study of the effect of the N-1 substituent on the photochemistry and phototoxicity of FQ antibiotics.These findings will be beneficial to the development of novel FQ antibiotics without phototoxicity.
文摘To identify the unknown proteins that would contaminate the α- and β-subunits of nitrogenase MoFe protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, the partially purified MoFe protein （Avl） preparation was obtained from Azotobacter vinelandii Lipmann OP by chroma- tography on DEAE-cellulose （DE52） and Sephacryl S-200 columns and analyzed by PAGE and matrix- assisted laser desorption/ionization time-of-flight （MALDI-TOF） mass spectrometry. The Av 1 preparation was shown to have two main bands at the position of the α- and β-subunits of crystalline Avl on the SDS gel. However, on the anoxic native PAGE, in addition to the Avl band, the preparation was shown to have three other main bands that migrated slower than Av 1. Of these three main bands, the protein with the fastest migration was identified as bacterioferritin elsewhere. The proteins on the other two bands, termed Upper and Middle, were suggested to be two different homopolymers with the same apparent subunit electrophoretic mobilities as the α- and β-subunits of Avl, respectively. By analysis of MALDI-TOF mass spectrometry, the Upper was identified as GroEL, which belongs to the heat shock protein 60 family, and the Middle was identified as glucose-6-phosphate isomerase （PGI）. In our preparation, anoxic native electrophoresis indicated that GroEL was composed of 14 identical subunits and that PGI was composed of 10 identical subunits. This is the first report of PGI, with so many subunits. The contaminating proteins in the Av 1 preparation, mainly GroEL and PGI, could be totally or partially removed from Av 1 if the shoulders and center of the elution peak were collected separately from the Sephacryl S-200 column and the center fraction was purified further by Q-Sepharose developed with an NaC1 concentration gradient. Thus, Avl with more than 90% purity was obtained. Obviously, this modified method is useful for the purification of mutant MoFe proteins with a high purity.
基金Supported by the State Key Basic Research and Development Plan of China （001CB1089-06） and the National Natural Science Foundation of China （30270296）.Acknowledgements The authors thank Professors YX Jing and JG Li for their valuable advice.
文摘A new protein, an approximately 59-kDa monomer containing iron atoms, was first isolated from the mutant strain DJ35 of Azotobacter vinelandii Llpmann. After analysis by matrix-assisted laser desorptlon ionization time-of-flight mass spectrometry, the protein was Identified as the product of a predicted gene. Thus, the protein was tentatively called HBP59. Its absorption spectra （ABS） In the reduced state exhibited three peaks at 421,517, and 556 nm and the maximal peak was shifted from 421 to 413 nm after exposure of HBP59 to air. The Soret circular dichrolsm （CD） spectrum of HBP59 In the reduced state displayed four positive peaks at 364, 382, 406, and 418 nm and two negative peaks at 398 and 433 nm; the △ε （CD extinction coefficient） values of these peaks were found to be 0.92, 0.58, 0.87, 0.72, -0.65 and -1.12 L/mol per cm, respectively. Titration with heme showed that the protein has 0.1 heme molecules/protein molecule. After HBP59 had fully Interacted with heme, Its maximal ABS value and Soret CD Intensity were increased by approximately 10-fold compared with values before Interaction. Therefore, It seems that one molecule of HBP59 can be interacted with only one heme. These results indicate that HBP59 contains heme with low spin and may be Involved In heme utilization or adhesion.
