diaminorhodamine-4m and 4-5-diaminofluorescein

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.

Topics: Cell Extracts; Fluorescein; Fungal Proteins; Hydrogen Peroxide; Indicators and Reagents; Intracellular Space; Nicotiana; Nitric Oxide; Peroxidase; Reactive Oxygen Species; Rhodamines; Spectrometry, Fluorescence

We studied the spontaneous increase in the fluorescence intensity of 4,5-diaminofluorescein. A slow, steady increase in fluorescence continued for at least 125 h, and this increase was accompanied by ca. 2 nm red shift in the peak of emission spectrum. The spontaneous increase also occurred to diaminorhodamine-4M and a fluorinated form of diaminofluorescein, which has been also used for the detection of nitric oxide (NO). We found that several factors (excitation light, pH etc.) did not alter the time course of this increase. Moreover, we found that this spontaneous increase can produce false-positive results when measuring low-rate nitric oxide production in human umbilical vein endothelial cells, and may confound the interpretation of results of NO production. We show that this adverse effect can be avoided by careful grouping of samples during measurement.

Topics: Fluorescein; Fluorescence; Fluorometry; Human Umbilical Vein Endothelial Cells; Humans; Indicators and Reagents; Nitric Oxide; Rhodamines