1998 Fiscal Year Final Research Report Summary
Development of HPLC-Fluorescence Determination method of NO and Its Application to Biological Samples
| Project/Area Number |
09672189
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Physical pharmacy
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| Research Institution | Nagasaki University |
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Principal Investigator |
NAKASHIMA Kenichiro Nagasaki University, School of Pharmaceutical Sciences, Professor, 薬学部, 教授 (30039656)
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| Co-Investigator(Kenkyū-buntansha) |
ARIZONO Koji Nagasaki University, Faculty of Environmental Studies, Associate Professor, 環境科学部, 助教授 (70128148)
KURODA Naotaka Nagasaki University, School of Pharmaceutical Sciences, Associate Professor, 薬学部, 助教授 (50234612)
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| Project Period (FY) |
1997 – 1998
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| Keywords | nitric oxide / high-performance liquid chromatography / 2,3-diaminonaphtalene / hemoglobins / 1(H)-2,3-naphthotriazole / cultivated plant cells / NO-production |
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| Research Abstract |
A high-performance liquid cliromatographic (HIPLC) determination of NO with fluorescence reaction product was confirmed The fluorescent reaction product was confirmed to be 1t(H)-2,3-naphthotriazole (NT) by an instrumental analysis. The peak on the chromatogram dcrived from the reaction product of SNAP with 2,3-diaminonaphthalene (DAN) was well corresponded to that of NT.Trapping abilities of some hemoglobins for NO were evaluated by using the developed method The order of trapping abilities of these compounds including dimethyldithiocarbamic acid (DDCT)-Fe complex, was DDTC-Fe >> oxyhemoglobin*myoglobin > hemoglobin >> methehemoglobin.
The present method was applied to the determination of NO in a cultured plant cells. After the incubation of cultured cells of Agave pacittica with DAN for 3h, cells were homogenized in methanol and centrifuged. Portions of 20 muI of the supernatant were injected onto HPLC.The detection limit of standard NO-DAN was 8.0 fmol on column (S/N=3). The incorporation rate of DAN into plant cells was about 19.6%, and the estimated NO concentration in cells was 5.3*1.6 pmol/g (n=3). A study on the role of NO in plant tissues is under investigation.
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