Purification of Pig Copper,Zinc-Superoxide Dismutase by Immobilized Metal Ion Affinity Chromatography
固定化金属离子亲和色谱法纯化猪铜锌超氧化物歧化酶

Copper,zinc superoxide dismutase (Cu,Zn-SOD,EC,1.15.1.1)catalyzes the dismutation of the superoxide radical produce O2,and avoid destroying organisms,Cu,Z-SOD has been used experimentally and clinically as a therapeutic drug.It is usually purified by precipitation with ammonium sulphate, ion -exchange chromatography and gel filtration. However the yields and extent of purification are relatively low.The development of immobilized metal ion affinity chromatography offers a new approach to enzyme purification.Iminodiacetic acid (IDA)was coupled to epoxy-activated Sepharose 4B.The column of IDA-epoxy-Sepharose 4Bwas activated by copper sulfate solution.Cu,Zn-SOD solution was applied to the column which had been equilibrated with phosphate buffer, then eluted with (1)buffers of phosphate and acetate containing imidazole and (2)buffers of phosphate and Tris-HCl, respectively.The results show that (1) high purification fold (38.4) with activity recovery over 90% for crude Cu,Zn-SOD solution using Cu2 -Sepharose 4B column was obtained,much more than those of DEAE-52 ionexhange column,(2)elution efficiency of the buffers of phosphate and Tris -HCl is better for the enzyme solution having more proteinic impurities, while the buffers of phosphate and acetate containing imidazole are fit for the enzyme solution having fewer proteinic impurities.