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

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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.