Affinity chromatography for the purification of glutathione S-transferase from Oxya chinensis

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1 * S - 2 ** GSH-agarose Oxya chinensis Thunberg 5 S - glutathione S-transferases GSTs 60% ~ 80% GSTs 90% μmol / min / mg protein. 82 GSH-agarose μmol / min / mg protein SDS- PAGE ku S - Affinity chromatography for the purification of glutathione S-transferase from Oxya chinensis GUO Yan-Qiong 2 WU Hai-Hua XUAN Tao ZHANG Jian-Zhen GUO Ya-Ping MA En-Bo **. Institute of Applied BiologyShanxi UniversityTaiyuan China 2. College of AgricultureShanxi Agriculture UniversityTaigu 03080China Abstract Glutathione S-transferase GST in the fifth-instar nymphs of Oxya chinensis Thunberg was purified by ammonium sulfate and GSH-agrose affinity chromatography. Higher specific activity of GSTs could be detected at 60% - 80% purityand the highest specific activity was found at 90%. The specific activity was μmol / min / mg protein and the purification factor was. 82-fold. Further GSH-agrose affinity chromatography indicated that purification reached with specific activity of μmol / min / mg protein. SDS-PAGE analysis indicated that the purified GSTs preparation had a single band with a relative molecular mass of ku. These results provide the basis for further study of the enzymatic propertiesstructural characteristics and functions of the GSTs of O. chinensis. Key words glutathione S-transferases GSTs Oxya chinensispurificationaffinity chromatography S- glutathione S-transferases GSTs Qin et al. 20 Oxya chinensis Thunberg Insecta Orthoptera Acridoidea Oedipodidae Oxya Serville S - * ** maenbo2003@ sxu. edu. cn

2 4 S % 30 min min g S - min GSTs 30% ~ 00% 4 0 min g 30 min S - 0. mol / L ph % Triton X- 00 GSTs GSTs 4 GSH-agrose GSTs GSH-agrose GSTs 60% ~ 90% GSTs 2 h NH + 4. GSH-agrose.. Oxya GSH-agrose chinensis 0 0. mol / L ph mol / L NaCl ml / 5 2 ~ 3 d min mmol / L ph % Triton X GSH-agrose Sigma 50mmol / L ph9. 0 Tris-HCl 8 mmol / L GSH GSH BBI GSTs CDNB GSTs BSABio-Rad GSTs Zhu He.. 3 DY89 - Ⅱ 2000 CDNB GSH 0 μl 90 μl Sigma DYZC-24DN 200 mmol / L GSH mmol / L CDNB SpectraMAX 90 GSH CDNB = nm Molecular Device 0 s min OD μl. 2. GSTs - 80 CDNB 9. 6 m mol L cm - mol / L ph % Triton X-00 0 W / V 5 Bio-Rad Protein 500 rpm 5 min Assay g 20 min GSTs GSTs SDS Smith 985 PAGE 5% GSTs 4% R - 250

3 GSTs GSTs 5 GSTs 60% ~ 90% GSTs 2 GSTs 3 90% μmol / GSTs min / mg μmol / min / mg. 82 GSTs GSTs 60% ~ 90% GSH-agrose GSH-agrose Fig. Fig. 2 GSH-agrose affinity chromatography of the GSTs from Oxya chinensis GSTs Specific activity of GSTs in different NH 4 2 SO 4 saturation 2 GSH-agrose GSTs Table GSTs GSH-agrose Purification results of the GSTs from Oxya chinensis with ammonium sulfate and GSH-agrose affinity chromatography Purification step GSTs GSTs Total enzyme GSTs specific Total protein Purification activity activity Yield % mg fold μmol / min μmol / min / mg Crude extract % ~ 90% Ammonium sulfate with saturation 60% 90% GSH-agrose GSH-agrose affinity chromatography

4 S - 829 GSTs GSTs 0. 674 μmol / min / mg 60% ~ 90% Qin GSTs 0. 3046 μmol / et al. 20 GSTs min / mg. 82 23. 70% GSH-agrose GSTs 8. 585 μmol / min / mg 50. 88 2009 8. 54% 2.

4 4 S GSTs GSTs μmol / min / mg 60% ~ 90% Qin GSTs μmol / et al. 20 GSTs min / mg % GSH-agrose GSTs μmol / min / mg % 2. 3 GSTs S - GSH-agrose 3 SDS GSTs 3 Chien Dauterman 99 Helicoverpa zea GSTs 4. 3 Yawetz 3 GSTs SDS-PAGE Fig. 3 SDS-PAGE electrophoretogram of tube 3 with GSH-agrose affinity chromatograph M Protein markers 2 Crude GSTs 3 Dialysate 4 Purified GSTs. GSTs Koren 984 Ceratitis capitata GSTs 6. 3 GSTs GSH-agrose GSTs 3 GSH-agrose 3 GSTs SDS-PAGE ~ ku GSTs 200 Kunanz SDS-PAGE Nanen 2004 GSH-agrose Tetranychus urticae 3 Rauch Nanen 2004 Bemisia tabaci ~ ku ku 3 GSH-agrose Locusta migratoria manilensis 3 S - S - S - 2 S -

5 S -. GSH-agrose 2 GSTs Qin GHJia MLiu TXuan TZhu KYGuo YPMa EB Zhang JZ20. Identification and characterization of ten glutathione S-transferase genes from oriental migratory locustlocusta migratoria manilensis Meyen. Pest GSTs Manag. Sci. wileyonlinelibrary. com DOI / ps. GSTs 85% Rauch NNauen R2004. Characterization and molecular GSTs cloning of a glutathione S-transferase from the whitefly 90% Bemisia tabaci Hemiptera Aleyrodidae. Insect Biochem. GSTs 24 ku Mol. Biol GSTs ku Smith PKKrohn RIHermanson GTMallia AKGartner GSTs ± 3. ku FHProvenzano MDFujimoto EKGoeke NMOlson BJ Klenk DC985. Measurement of protein using 2009 bicinchoninic acid. Anal. Biochem S - GSH-agrose GSTs S S References Chien CDauterman WC99. Studies on glutathione S transferase in Helicoverpa = Heliothis zea. Insect Yawetz AKoren B984. Purification and properties of the Biochemistry Mediterranean fruit fly Ceratitis capitata W. glutathione S S - transferase. Insect Biochemistry Kunanz SNauen R2004. Purification and partial characterization of a glutathione S-transferase from the twospotted spider mitetetranychus urticae. Pestic. Biochem. Phys S Zhu KYHe FQ2000. Elevated esterases exhibiting arylesterase-like characteristics in an organophosphateresistant clone of the greenbugschizaphis graminum Pestic. Biochem. Phys GST

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