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CL assay of ALP using dihydroxyacetone phosphate as substrate with lucigenin
Amane Kokado*; H Arakawa; M Maeda
Sch. of Pharm. Sci., Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo, 142-8555, JAPAN *(email@example.com)
Lucigenin produces strong chemiluminescence (CL) with hydrogen peroxide or reducing agents. Previously, we have reported that many compounds having an alpha- hydroxycarbonyl group in the side chain reacts with lucigenin to generate CL. In the reaction of lucigenin with various reducing compounds, dihydroxyacetone (DHA) gave the most intense CL. Therefore, we reported the CL assay of alkaline phosphatase (ALP) using glycerol 3-phosphate or NADP+ as substrate and glycerol dehydrogenase as the coupled enzyme (M. Kitamura, M. Maeda and A. Tsuji, J. Biolum. Chemilum., 10 (1995) 1).
We present here the development of more simple detection method for ALP using dihydroxyacetone phosphate (DHAP) as a new substrate. ALP hydrolyzed DHAP to form DHA, which reacts with lucigenin to emit light. In the optimum assay condition, the detection limit of ALP was 0.38 amol. The CL mechanism of lucigenin with DHA was investigated by ESR spectrometry using spin trapping method. The mechanism was speculated as follows: the O2- generated by the reaction of DHA and O2 in alkaline solution react with lucigenin, and emit light. We also applied this assay to enzyme immunoassay using ALP as label.
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