Hydropropidine: a novel, cell-impermeant fluorogenic probe for detecting extracellular superoxide.
Here we report the synthesis and characterization of a membrane-impermeant fluorogenic probe, hydropropidine (HPr(+)), the reduction product of propidium iodide, for detecting extracellular superoxide (O(2)(•-)). HPr(+) is a positively charged water-soluble analog of hydroethidine (HE), a fluorogenic probe commonly used for monitoring intracellular O(2)(•-). We hypothesized that the presence of a highly localized positive charge on the nitrogen atom would impede cellular uptake of HPr(+) and allow for exclusive detection of extracellular O(2)(•-). Our results indicate that O(2)(•-) reacts with HPr(+) (k=1.2×10(4) M(-1) s(-1)) to form exclusively 2-hydroxypropidium (2-OH-Pr(2+)) in cell-free and cell-based systems. This reaction is analogous to the reaction between HE and O(2)(•-) (Zhao et al., Free Radic. Biol. Med.34:1359-1368; 2003). During the course of this investigation, we also reassessed the rate constants for the reactions of O(2)(•-) with HE and its mitochondria targeted analog (Mito-HE or MitoSOX Red) and addressed the discrepancies between the present values and those reported previously by us. Our results indicate that the rate constant between O(2)(•-) and HPr(+) is slightly higher than that of HE and O(2)(•-) and is closer to that of Mito-HE and O(2)(•-). Similar to HE, HPr(+) undergoes oxidation in the presence of various oxidants (peroxynitrite-derived radicals, Fenton's reagent, and ferricytochrome c) forming the corresponding propidium dication (Pr(2+)) and the dimeric products (e.g., Pr(2+)-Pr(2+)). In contrast to HE, there was very little intracellular uptake of HPr(+). We conclude that HPr(+) is a useful probe for detecting O(2)(•-) and other one-electron oxidizing species in an extracellular milieu.
Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA. rmichalski@mcw.edu
Radoslaw
R
Michalski
Jacek
J
Zielonka
Micael
M
Hardy
Joy
J
Joseph
Balaraman
B
Kalyanaraman
D000818:Animals; D001693:Biological Transport, Active; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D005110:Extracellular Space / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D016877:Oxidants / Q000378:metabolism; D010084:Oxidation-Reduction; D010617:Phenanthridines / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011419:Propidium / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000138:chemical synthesis / Q000378:metabolism*; D012995:Solubility; D013481:Superoxides / Q000378:metabolism*
D005456:Fluorescent Dyes; D016877:Oxidants; D010617:Phenanthridines; D000644:Quaternary Ammonium Compounds; C582101:hydropropidine; D013481:Superoxides; D011419:Propidium; C058428:hydroethidine