两种酶谱方法在κ-卡拉胶酶活性鉴定中的应用

Application of Two Zymography in κ-carrageenase Activity Identification

对分离纯化后的κ-卡拉胶酶进行SDS-PAGE电泳和酶谱试验鉴定,比较κ-卡拉胶酶活性鉴定中的两种酶谱试验方法。一种是先进行非变性PAGE电泳,电泳完毕,将电泳胶与事先准备好的底物胶叠合在一起,35℃孵育液孵育。另一种是在此试验方法基础上进行改进,直接在电泳分离胶中加入0.2%底物,进行SDS-PAGE电泳,电泳结束,用TritonX-100将电泳胶复性,孵育液孵育。试验结果表明改进后的酶谱方法操作简单,具有良好的灵敏度和精确的定位。同时,利用改进后的酶谱方法对κ-卡拉胶酶活性进行了反应时间的研究,结果显示,反应时间为8 h时,降解条带最清晰,最有利于相似分子量酶的辨别。

After purification, the κ-carrageenase activity was identified by the sodium dodecyl sulfate polyacrylamide gel electrophoresis （ SDS-PAGE ） and zymography experiment. Both zymography methods were compared in this report. One zymography experiment, firstly, diseontinuous native PAGE was carried out to separate κ-earrageenase. After eleetrophoresis, remove the separating gel, carrageenase activity was detected by overlaying another κ-carrageenan-eontaining gel （ 5% acrylamide, pH 6.8, 0.2% carrageenan, preequilibrated in Na2HPO4- citric acid buffer [ pH 7.0 ] ）. The gels were then incubated together at 35℃ . Compared with the former method, the other method has made few improvements. SDS-PAGE was performed in a separating gel with 0.2% K-carrageenan, after eleetrophoresis, the separating gel with 0.2% ^-carrageenan was rinsed with TritonX-100 solution repeatedly, and the gel was then incubated in incubation （ 0.05 mol/L Tris-HC1, 0.2 mol/L NaCI, 0.01 mol/L CaC12, 1% TritonX-100, pH7.0 ） at 35~C. Experimental results showed that the improved zymogram method is simple to operate, with satisfactory sensitivity and precision positioning. Simultaneously, the reaction time was studied for κ-carrageenase activity using the improved zymography methods. It was suggested that 8 h is the optimal incubation time, at this moment, with the dearest light bond and most beneficial to identify similar molecular mass enzyme.