核糖体脱嘌呤分析法分析检测核糖体失活蛋白

Ribosome Depurination Assay for Ribosome Inactivating Protein Purification

针对核糖体脱嘌呤分析法经常被用来分析检测新的核糖体失活蛋白(RIP),检测了由酸性苯胺催化在RIP催化脱嘌呤位点断裂产生的核糖体rRNA片段。利用文献发表的一级动力学速度常数,计算分析了核糖体失活蛋白含量对核糖体失活蛋白催化脱嘌呤分析的敏感性,并进行了部分实验验证。实验中核糖体失活蛋白和核糖体溶液混合后进行了30 min的恒温培养,结果表明:核糖体失活蛋白脱嘌呤分析法的敏感性取决于核糖体失活蛋白催化核糖体脱嘌呤反应的速度常数。还分析了核糖体失活蛋白脱嘌呤分析中可能出现的假阳性和假阴性结果及其原因,结果表明:在核糖体失活蛋白的大规模生产中使用核糖体脱嘌呤分析法作为定量分析的手段是比较复杂和困难的,该方法应该和一些常用的经典方法例如SDS PAGE分析法联合使用,而且联合使用时两种方法的最小检测限应该相近,以避免其中之一引起的假阳性结果。

The ribosome depurination assay has been frequently used to identify new ribosome inactivating proteins （RIPs）. The assay measures a ribosomal rRNA fragment generated by acidic analine catalyzed cleavage at the RIP catalyzed depurination site. Using published first order kinetic rate constants for RIP catalyzed depurination, the sensitivity of the depurination assay to RIP concentration has been determined. For a 30 minute incubation time of the RIP with a ribosome containing solution, the sensitivity of the depurination assay depends on the rate constant for the RIP catalyzed ribosome depurination. In addition common sources of false positive and false negative results have been determined. These results indicate that use of the depurination assay as a quantitative analytical method in the commercial manufacture of RIPs is complex. In addition if the depurination assay is to be combined with a well established method such as SDSPAGE, it is important that the minimum detection levels of the two methods be similar in order to prevent a positive result by one method and not the other.