Development of HPLC-Fluorescence Determination method of NO and Its Application to Biological Samples

点击翻译按钮获取中文摘要

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.