猪小肠粘膜碱性磷酸酶的分离纯化及性质研究

Purification and Characterization of Alkaline Phosphatase in Porcine Intestinal Mucosa

猪小肠粘膜经过Tris-HCl缓冲液(pH8.5)充分溶解、正丁醇除脂、冷冻离心、真空冻干等步骤处理后,先后经过Phenyl High Performance疏水层析/DEAE Fast Flow阴离子层析/Sephacryl S-200凝胶层析纯化后得到猪小肠粘膜ALP(碱性磷酸酶),并对其蛋白二级结构及理化性质进行研究。结果表明:该酶经过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离得到ALP蛋白带,分子量约为57.0 kDa。该酶纯化倍数为68.2,比活力倍数为19.1 U/mg。利用圆二色性光谱测定纯化后蛋白二级结构,发现α-螺旋3.6%、β-折叠41.8%、β-转角21.5%、无规则卷曲33.1%。ALP在催化底物对硝基苯磷酸二钠(p-NPP)作用下最佳温度30℃、最佳pH为9.5;金属离子对ALP起到激活的离子为Mg^2+和Ca^2+,起到抑制的离子为Zn^2+和EDTA。

The pig intestinal mucosa was fully dissolved in Tris-HCl buffer(pH8.5),degreased with n-butanol,frozen and centrifuged,and lyophilized in vacuum.Phenyl high performance hydrophobic chromatography,DEAE fast flow anion chromatography,Sephacryl S-200 gel chromatography were used to purified and the porcine small intestinal mucosa ALP(alkaline phosphatase)was obtained.Secondary structure and physicochemical properties of the ALP were determined.The results showed that the ALP protein band was separated by sodium lauryl sulfate-polyacrylamide gel electrophoresis(SDSPAGE),and the molecular weight was about 57.0 kDa.The purification factor of ALP was 68.2,and the specific activity factor was 19.1 U/mg.The secondary structure of the protein was determined by circular dichroism spectroscopy,and the results showed that the protein was-helix 3.6%,section-fold 41.8%,section-corner 21.5%,and irregular curl 33.1%.The enzyme catalyzed the substrate disodium nitrophenylphosphate(p-NPP)with an optimal temperature of 30℃ and an optimal pH value of 9.5.Metal ions Mg^2+ and Ca^2+ could activate ALP,and the inhibition ions were Zn^2+ and EDTA.