Development of a flow type of current application measuring system armed with specific capture and detectability for heavy metal ions

在第一年中，使用螯合剂再生脱辅基酶的化学方法应用于流动注射分析，并与基于电解的方法进行比较。用吡啶二羧酸和EDTA两种螯合剂对脱辅基酶进行再生。我们发现前者能有效地灭活大肠杆菌的碱性磷酸酶，而后者暴露于小牛肠的ALP则能有效地获得脱辅基酶。再生和再活化脱辅基酶之间的交替转换过程的详细调查表明，PDC应吸附到固定在多孔玻璃珠上的酶的表面上，从而，协调的酶的活性中心的底物应被阻止。在第二年，将N-二甲基羧基肌氨酸（DTCS）作为钴（II）离子的掩蔽剂加入到样品金属溶液中，然后进样到一次螯合剂暴露的柱中。因此，证实了可以观察到归因于锌（II）离子的酶柱的再活化。总之，选择性微量测定锌（II）离子可以实现通过使用掩蔽剂。此外，过量的锌（II）离子吸附到CPG检测通过监测从酶柱的流出物。因此，将来自大肠杆菌的ALP固定在网状金上，而不是使用CPG作为支持材料，从而可以显著改善归因于脱辅基酶柱的再活化的酶活性的测定的再现性。这些结果可为真实的样品中重金属离子的微量分析提供重要信息。

In the first year, a chemical method for regenerating the apoenzymes with use of chelating agents was applied to the flow-injection analysis and compared with a method based on electrolysis. Two kind of chelating agents such as pyridine dicarboxylate and EDTA were used for regeneration of the apoenzymes. We found that the former efficiently deactivated alkaline phosphatase from E.coli, and that exposure of the latter to the ALP from calf intestine was effective for gaining the apoenzymes. Detail investigation on the alternate conversion processes between regenerating and reactivating apoenzymes demonstrated that PDC should be adsorbed onto the surface of the enzymes immobilized onto the porous glass beads and thereby, coordination to the active center of the enzyme by the substrates should be prevented. Therefore, the enzymes were apparently inhibited by addition of PDC solution.In the second year, N-dimethylcarboxylsarcosine (DTCS) as masking agents for cobalt(II) ions was added to the sample metal-solutions and then, injected into the once chelator-exposed column. Thus, it was confirmed that reactivation of the enzyme column attributable to zinc(II) ions could be observed. In conclusion, selective microdetermination of zinc(II) ions could be achieved by using the masking agent. Furthermore, excess amount of zinc(II) ions adsorbed onto the CPG was detected by monitoring the effluents from the enzyme column. Therefore, ALP from E.coli was immobilized onto a net type of gold instead of using CPG as the supporting material and thereby, reproducibility of the assay for the enzyme activity attributable to reactivation of the apoenzyme column could be significantly improved. These results can provide crucial information to microanalysis of heavy metal ions in real samples.