摘要
先用50mmol／LpH7．6磷酸盐缓冲液（PBS）提取，再经55％～60％（质量分数）硫酸铵盐析分级分离、冷冻乙醇一氯仿沉淀除杂和丙酮再沉淀分级由成熟的K326鲜烟叶得到含CuZn-SOD的粗酶液，粗酶液经DEAE．52柱层析（20mmol／LPBS平衡洗脱6h，流速0．25mL／min；600mL 20mmol／LPBS和600mL 100mmol／LPBS线性梯度洗脱，流速0．25mL／min）和DEAE-52离子交换柱层析（30～100mmol／LPBS线性梯度洗脱，流速0．25mL／min），成功将3种CuZn-SOD同工酶分开；再通过G．75凝胶柱层析（10mmol／LpH7．6PBS洗脱，流速0．3mL／min）和-20℃下冷冻干燥，得到CuZnSODⅠ和CuZnSODⅢ纯品，经测定，CuZnSODI由302个氨基酸残基组成，其相对分子质量为36682Da，最大吸收波长262nm，最适pH6．0左右，最适温度40℃；CuZnSODⅢ含187个氨基酸残基，相对分子质量为22976Da，最大吸收波长269nm，最大荧光激发波长和发射波长分别为280和338nm，最适pH5．0～6．0，最适温度30℃。
Three isoenzymes, Cu, Zn-superoxide dismutases Ⅰ, Ⅱ and Ⅲ, were isolated from ripe fresh tobacco leaves （K326,Nicotinana） by extraction with 50 mmol/L, pH7.6 phosphate buffer solution （PBS）, successive precipitation with 55% -60% （w/w） ammonium sulfate, frozen ethanol-chloroform and acetone, and isolation by DEAE-52 column （balance eluted for 6h with 20mmol/L, pH 7.6 PBS, flow rate 0.25mL/min, then linear gradient-eluted with 20 - 100mmol/L PBS, flow rate 0.25mL/min） and then by DEAE-52 ion-exchange column （linear gradient-eluted with 30-100 mmol/L PBS, flow rate 0.25mL/min）. Pure Cu, Zn-superoxide dismutases Ⅰ and Ⅲ were prepared further by G-75 gel column （eluted with 10mmol/L, pH7.6 PBS, flow rate 0.3 mL/ min） and freeze-dried at -20℃. It was found that Cu, ZnSOD Ⅰ consisted of 302 amino,acid residues, its relative molecular mass was 36682 Da, maximum absorption wavelength was 262 rim, optimal pH value was around 6.0, and optimal temperature was 40℃ ; Cu, ZnSOD Ⅲ consisted of 187 amino acid residues, its relative molecular mass was 22976 Da, maximum absorption wavelength was 269 nm, the maximum fluorescence excitation and emission wavelengths were 280 nm and 338 nm,respectively, optimal pH value was 5.0 -6.0, and optimal temperature was 30℃.
出处
《烟草科技》
EI
CAS
北大核心
2007年第4期40-44,61,共6页
Tobacco Science & Technology
