| Project/Area Number |
06660137
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Bioproduction chemistry/Bioorganic chemistry
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| Research Institution | FUKUI PREFECTURAL UNIVERSITY |
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Principal Investigator |
SENDA Mitsugi FUKUI PREFECTURAL UNIVERSITY,DEPARTMENT OF BIOSCIENCE,PROFESSOR, 生物資源学部, 教授 (90026419)
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| Co-Investigator(Kenkyū-buntansha) |
YAMAGUCHI Seiki FUKUI PREFECTURAL UNIVERSITY,DEPARTMENT OF BIOSCIENCE,PROFESSOR, 生物資源学部, 教授 (90084897)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1994: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | electroanalytical chemistry / enzyme activity assay / catalytic current / enzyme cycling / 4-aminophenol / diaphorase / substrate inhibition / alkaline phosphatase |
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| Research Abstract |
A high-sensitive electrochemical method of the determination of a mediator (Med) based on the catalytic oxidation current of NADH involving mediator (Med) /diaphorase (Dia) /NADH system and its application to an electrochemical method of enzyme assay have been studied. (1) Electrochemical as well as spectrochemical studies have shown that the enzymatic reaction Med/Dia/NADH proceeds according to the sequential mechanism when the Med is a 1-electron redox couple, like hexacyanoferrate (-3/-4) and ferrocene (+1/0), whereas it proceeds according to the ping-pong mechanism when the Med is 2-electron redox couple, like DCIP.(2) The behavior of the catalytic current can be explained by the basic equation of the Med/Dia/NADH system, when the concentration of Med is low enough, independently of the enzymatic reaction mechanism. (3) The Med/Dia/HADH reaction system has been proved to be interfered by substrate (NADH) -inhibition. (4) The enzymatic reaction velocity equations for both competitive and non-competitive inhibitions have been derived and applied to analyze the catalytic currents inhibited by NADH,giving the effective inhibitor constants. (5) On the basis of these results, the optimum concentration of HADH has been determined to be 0.5mM to design the electrochemical method for trace determination of Med ; under this newly established condition a mediator, 4-aminophenol, can be determined at the concentration level as low as 5x10^<-11> M.(6) The electrochemical method can be applied to assay alkaline phosphatase activity, in which the hydrolysis of 4-aminophenophosphate by alkaline phosphatase is followed electroanalytico-chemically.Alkaline phosphatase in the concentration level as low as 5x10^<-8> units/50mul can be determined. (7) The result shows that a high-sensitive electrochemical enzyme immune assay method based on the present electrochemical method of alkaline phosphatase assay is promising.
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