铜螯合亲和层析分离抗氧化活性蚕豆蛋白酶解物

摘要

Immobilized metal affinity chromatography (IMAC) was used to separate broad bean protein hydroly sates with different copper binding ability,the relationship between antioxidant activity and the copper binding ability of broad bean protein hydrolysates was discussed as well.The results showed that the optimal conditions for metal affinity chromatography were balance buffer,0.05 mol/L sodium acetate-acetic acid (NaAc-HAc) buffer at pH 5.0,loading sample:1 mL of broad bean protein hydrolysate solution (20 mg/mL),elution buffers,0.04 mol/L imidazole.After being eluted,F1 (the broad bean hydrolysates component not binding to copper column) and F2 (component binding to copper column) were separated.Furthermore,copper binding abilities and antioxidant activities of broad bean protein hydrolysates,F1 and F2 were investigated.The results were shown F2 ＞ the broad bean protein hydrolysates ＞F1 (P ＜0.05) in both copper binding abilities and antioxidant activities.These results presented that the stronger copper binding ability of the broad bean protein hydrolysate,the higher antioxidant activities.%采用铜离子螯合亲和层析柱对不同铜螯合能力的蚕豆蛋白酶解物组分进行分离,并探讨其抗氧化活性机制与铜螯合能力之间的关系.结果表明,金属亲和层析的最优条件为:平衡缓冲液为pH 5.0,浓度为0.05 moL/L的NaAc-HAc;上样量为20 mg/mL的蚕豆蛋白酶解物1 mL;洗脱剂为0.04 mol/L咪唑.洗脱得到不能螯合铜的蚕豆蛋白酶解物组分F1及能螯合铜的组分F2,测定其总还原力、抑制羟自由基能力与铜螯合量,发现抗氧化活性的关系为F2＞蚕豆蛋白酶解物＞ F1 (P ＜0.05),铜螯合量的关系也为F2＞蚕豆蛋白酶解物(未经分离)＞ F1(P ＜0.05),表明蚕豆蛋白酶解物的铜螯合活性越高,其抗氧化活性越高.