1992 Fiscal Year Final Research Report Summary
Superoxide measurement system by pH-jump method
| Project/Area Number |
03671040
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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 |
Physical pharmacy
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| Research Institution | Tokyo Metropolitan Institute of Medical Science |
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Principal Investigator |
FUJII Hirotada Tokyo Metropolitan Institute of Medical Science, 炎症, 研究員 (70209013)
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| Co-Investigator(Kenkyū-buntansha) |
KAKINUMA Katsuko Tokyo Metropolitan Institute of Medical Science, 炎症, 研究員 (30109946)
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| Project Period (FY) |
1991 – 1992
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| Keywords | Superoxide / Active oxygen / pH-Jump / ESR / Nitroso compound |
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| Research Abstract |
For direct measurement of the ESR signal of superoxide anion (O^-_) produced in biological. O^-_generated at a physiological pH was trapped in alkaline media instead of by a rapid freezing method, and then its signal was measured by ESR spectroscopy at 77K.A reaction mixture for O^-_generation, such as neutrophils and NADPH oxidase in neutrophils, was incubated at a physiological pH (pH7.0-7.5) for a suitable reaction period (30 s), then an aliquot (300 mul) was pipetted out and squirted into 600 mul of 0.5 M NaOH to stabilize O^-_(pH-jump). The alkaline mixture was promptly introduced into an ESR tube and frozen by dipping the tube directly into a cooling liquid. A typical signal of O^-_ was detected by ESR spectroscopy and the amount of trapped O^-_ was measured quantitatively at 77 K.
Based on this pH-jump method, we developed the superoxide measuring system which is controlled by the personal computer.
This system should be widely used for direct measurement of O^-_ produced in biological system at physiological pH.
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