Lewis B L, McGuinness E T
Department of Chemistry, Seton Hall University, South Orange, New Jersey 07079.
Anal Biochem. 1990 Jan;184(1):104-10. doi: 10.1016/0003-2697(90)90020-a.
NADP(H) measurements by enzymatic amplification are described in which the interface step between cycling (glucose-6-phosphate and glutamic dehydrogenases) and indicator (6-phosphogluconic dehydrogenase) enzymes has been reconfigured, permitting the entire operation to run as a continuous assay on a centrifugal fast analyzer. This is accomplished by using the sequential load feature of the analyzer and incorporating either sodium dodecyl sulfate (SDS) or SDS and hydrogen peroxide as kill reagents to replace the thermal step (destruction of cycle enzymes by boiling). The ability of SDS to render a cycle inoperative during the run time of the indicator enzyme depends on the inherent resistivity and absolute amount of its enzyme proteins to this surfactant. Criteria used to judge the efficacy of a potential kill reagent are based on the sample blank time-response curve and the cycle product recovery by the indicator enzyme. Various other enzyme cycling systems which can be fitted to the centrifugal fast analyzer are highlighted.
描述了通过酶促扩增进行的NADP(H)测量方法，其中循环酶（葡萄糖-6-磷酸脱氢酶和谷氨酸脱氢酶）与指示酶（6-磷酸葡萄糖酸脱氢酶）之间的界面步骤已重新配置，使整个操作能够在离心快速分析仪上作为连续测定运行。这是通过使用分析仪的顺序加载功能并加入十二烷基硫酸钠（SDS）或SDS和过氧化氢作为灭活试剂来取代热步骤（通过煮沸破坏循环酶）来实现的。SDS在指示酶运行期间使循环无效的能力取决于其酶蛋白对该表面活性剂的固有抗性和绝对量。用于判断潜在灭活试剂有效性的标准基于样品空白时间响应曲线和指示酶的循环产物回收率。还强调了可以适配到离心快速分析仪上的各种其他酶循环系统。
