小麥草超氧化歧化酶之純化及性質分析

Abstract

本研究針對小麥草汁中所含超氧化歧化酶進行性質分析，並比較純化前後之差異。小麥草汁之粗酵素液，經由熱處理、硫酸銨劃分以及離子交換層析等連續系列步驟，結果發現，可使超氧化歧化酶之純度提高約143.4倍，酵素活性回收率約為45.3%。將純化之酵素通入Superose 12膠體過濾層析測得原態分子量為46 kDa，SDS-PAGE經活性染色測得次單元分子量為23 kDa，因此為一同質性二元體酵素。PhastGel IEF膠片等電焦集電泳經活性染色分析結果顯示酵素之等電點（pI值）為3.95，屬酸性超氧化歧化酶。再經native-PAGE電泳及活性染色鑑定出膠體所顯示之酵素活性帶對KCN（8 mM）及H2O2（8 mM）兩種試劑皆不敏感，屬於Mn-SOD。小麥草純化後之超氧化歧化酶在pH 8時較穩定，而其粗酵素液中之超氧化歧化酶於20-30℃保溫60分鐘或40℃保溫15分鐘皆很穩定，活性幾乎無喪失，於40℃下靜置60分鐘活性始明顯降低，殘留活性約為75%。而純化之超氧化歧化酶於20-50℃保溫60分鐘，殘留活性為69~82%之間，然當溫度提高至60℃時，活性開始明顯降低，於60℃下靜置至60分鐘時僅存約16%。由殘留酵素活性百分率之對數值對保溫靜置時間作圖發現，小麥草超氧化歧化酶之熱失活反應在反應初期為一級反應，且溫度對熱失活速率之影響符合阿瑞尼亞方程式(Arrhenius equation)，低溫時粗酵素液之熱失活活化能為7.4×104 J/mol；而純化酵素液之熱失活活化能則為1.5×104 J/mol。化學修飾劑diethyl pyrocarbonate （2.5 mM）及Woodward,s reagent K.（50 mM）之抑制作用最強，可使酵素顯著的失去活性。因此組胺酸咪唑基（imidazole group）及天門冬胺酸或麩胺酸羧基（carboxyl group）可能為構成酵素活性中心所必須或位於其附近。

This study aimed at characterizing the superoxide dismutase from wheat grass before and after purification. Superoxide dismutase was purification from wheat grass, by sequential steps of heat treatment, ammonium sulfate fractionation and ion-exchange chromatography. It was found that by these steps, the purity of the superoxide dismutase increased by 143.4 fold and recovery of the enzyme activity was 45.3 %. The molecular mass of the superoxide dismutase from wheat grass was about 46 kDa, as estimated by Superose 12 gel filtration. The molecular mass of the subunit was 23 kDa, as determined by SDS-PAGE and activity staining. Thus, the enzyme is an homodimer containing 2 subunits. As estimated by isoelectrofocusing electrophoresis on PhastGel IEF 3-9 gel and zymogram staining, the isoelectric points ( pI ) of the superoxide dismutase was 3.95, indicating that the superoxide dismutase from wheat grass belonged to acid superoxide dismutase. The purified superoxide dismutase showed an apparent activity band , as determined by native-PAGE and gel activity staining with different type SOD inhibitors. The enzyme was Insensitive to cyanide ( 8 mM ) and hydrogen peroxide ( 8 mM ) . It belongs to Mn-SOD. The purified superoxide dismutase from wheat grass was stable at pH 8﹒The crude extract of superoxide dismutase from wheat grass was thermally stable after holding at 20-30℃ for 60 min or at 40℃ for 15 min, nevertheless the superoxide dismutase activity started to drop significantly after holding at 40℃ for 60 min.The residual superoxide dismutase activity was about 75%. Similarly, the purified superoxide dismutase from wheat grass was thermally stable after holding at 20-50℃ for 60 min. Approximately , 69~82 % enzyme activity was retained. However, as the temperature was higher than 60℃, the enzyme activity decreased significantly. The residual superoxide dismutase activity was only about 16% after holding at 60℃ for 60 min. The thermal inactivation reaction of superoxide dismutase during initial thermostat stage followed first-order reaction kinetics, and the temperature dependence of rate constants was in agreement with the Arrhenius equation. The activation energy of thermoinactivation of Low Temperature the crude extract and the purified superoxide dismutase from wheat grass was found to be 7.4×104 J/mol and 1.5×104 J/mol, respectively. Chemical modification agents, diethyl pyrocarbonate ( 2.5 mM ), Woodward''s reagent K ( 50 mM ) significantly inhibited the activity of the enzyme. Therefore, imidazole(histidine) and carboxyl (aspartic glutamic acid) groups are probably located at or near the active site of the enzyme.