6-O-季铵化壳聚糖修饰超氧化物歧化酶的制备及性质研究

Preparation and Characterization of Superoxide Dismutase Chemically Modified by 6-O-Quaternized Chitosan

目的用6-O-季铵化壳聚糖对超氧化物歧化酶(SOD)进行化学修饰,并初步分析修饰酶的理化性质。方法将2,3-环氧丙基三甲基氯化铵(GTMAC)接枝到壳聚糖的C6-OH上得到水溶性明显改善的O-(羟基)丙基-3-三甲基氯化铵壳聚糖(OHTCC)衍生物;通过碳二亚胺/N-羟基琥珀酰亚胺(EDC/NHS)活化法将O-(羟基)丙基-3-三甲基氯化铵壳聚糖连接到超氧化物歧化酶的羧基上,采用DEAE Sepharose Fast Flow弱阴离子交换柱色谱分离纯化得到结合物;十二烷基硫酸钠聚丙烯酰胺凝胶电泳法、高效液相色谱法及圆二色谱分析结合物的性质,邻苯三酚法测定酶活性。结果超氧化物歧化酶成功地被O-(羟基)丙基-3-三甲基氯化铵壳聚糖修饰,修饰率为90%,分离纯化得到的产物为连续分布的均一组分,修饰酶的活性保持率达到81.1%。结论 O-(羟基)丙基-3-三甲基氯化铵壳聚糖修饰超氧化物歧化酶的效率较高,且修饰酶的活性保持率较高。

OBJECTIVE To chemically modify superoxide dismutase（ SOD） with 6-O-quaternized chitosan. METHODS 2,3-Epoxypropyl trimethyl ammonium chloride was grafted to reactive C6-OH of chitosan to obtain O-（ 2-hydroxyl） propyl-3-trimethyl ammonium chitosan chloride（ O-HTCC）. O-HTCC was covalently coupled to SOD via EDC / NHS-mediated reaction,followed by purification on DEAE Sepharose fast flow column. The conjugates were analyzed by SDS-PAGE,HPLC,and circular dichroism. The enzyme activity of the conjugates was determined using pyrogallol autoxidation assay. RESULTS SOD was successfully modified by O-HTCC at an efficiency of 90%. The homogeneous conjugates showed similar secondary structure and retained 81. 1% of enzyme activity compared with native SOD. CONCLUSION SOD can be efficiently modified by O-HTCC to prepare O-HTCC-SOD conjugates with high activity retention rate.