University of Wuerzburg, Julius-von-Sachs Institute of Biosciences, Julius-von-Sachs-Platz 2, D-97082 Wuerzburg, Germany.
Nitric Oxide. 2012 Aug 15;27(2):123-35. doi: 10.1016/j.niox.2012.05.007. Epub 2012 Jun 6.
Diaminofluorescein-dyes (DAFs) are widely used for visualizing NO· production in biological systems. Here it was examined whether DAF-fluorescence could be evoked by other means than nitrosation. Tobacco (Nicotiana tabacum) suspension cells treated with the fungal elicitor cryptogein released compound(s) which gave a fluorescence increase in the cell-free filtrate after addition of DAF-2 or DAF-FM or DAR-4M. DAF-reactive compounds were relatively stable and identified as reaction products of H(2)O(2) plus apoplastic peroxidase (PO). CPTIO prevented formation of these products. Horseradish-peroxidase (HR-PO) plus H(2)O(2) also generated DAF-fluorescence in vitro. Using RP-HPLC with fluorescence detection, DAF derivatives were further analyzed. In filtrates from cryptogein-treated cells, fluorescence originated from two novel DAF-derivatives also obtained in vitro with DAF-2+HR-PO+H(2)O(2). DAF-2T was only detected when an NO donor (DEA-NO) was present. Using high resolution mass spectrometry, the two above-described novel DAF-reaction products were tentatively identified as dimers. In cells preloaded with DAF-2 DA and incubated with or without cryptogein, DAF-fluorescence originated from a complex pattern of multiple products different from those obtained in vitro. One specific peak was responsive to exogenous H(2)O(2), and another, minor peak eluted at or close to DAF-2T. Thus, in contrast to the prevailing opinion, DAF-2 can be enzymatically converted into a variety of highly fluorescing derivatives, both inside and outside cells, of which none (outside) or only a minor part (inside) appeared NO· dependent. Accordingly, DAF-fluorescence and its prevention by cPTIO do not necessarily indicate NO· production.
二氨基荧光素（DAFs）广泛用于生物系统中 NO·产生的可视化。在这里，研究了 DAF-荧光是否可以通过硝化成以外的其他方式来激发。用真菌诱导剂 cryptogein 处理的烟草（Nicotiana tabacum）悬浮细胞释放出化合物，这些化合物在添加 DAF-2 或 DAF-FM 或 DAR-4M 后，在无细胞滤液中引起荧光增加。DAF 反应性化合物相对稳定，并被鉴定为 H（2）O（2）加质外体过氧化物酶（PO）的反应产物。CPTIO 阻止了这些产物的形成。辣根过氧化物酶（HR-PO）加 H（2）O（2）也在体外产生 DAF 荧光。使用具有荧光检测的反相高效液相色谱法，进一步分析了 DAF 衍生物。在 cryptogein 处理的细胞的滤液中，荧光来自于两种新型的 DAF 衍生物，这两种衍生物也在体外与 DAF-2+HR-PO+H（2）O（2）一起获得。只有当存在 NO 供体（DEA-NO）时，才会检测到 DAF-2T。使用高分辨率质谱法，初步鉴定上述两种新型 DAF 反应产物为二聚体。在预先用 DAF-2 DA 加载并孵育的细胞中，无论是否有 cryptogein，DAF 荧光都源于与体外获得的不同的多种产物的复杂模式。一个特定的峰对外部 H（2）O（2）有反应，另一个较小的峰在 DAF-2T 处或接近洗脱。因此，与普遍观点相反，DAF-2 可以在细胞内外转化为多种高荧光衍生物，其中没有一种（在细胞外）或只有一小部分（在细胞内）表现出对 NO·的依赖性。因此，DAF 荧光及其被 cPTIO 抑制并不一定表示 NO·的产生。
