Dihydrotetramethylrosamine: A long wavelength, fluorogenic peroxidase substrate evaluated in vitro and in a model phagocyte

Abstract

Dihydrotetramethylrosamine, a fluorogenic substrate for peroxidase, and its fluorescent oxidation product, tetramethylrosamine chloride, were evaluated. The substrate is colorless and nonfluorescent while the oxidized dye absorbs at 550 nm and emits at 574 nm in both methanol and water. In vitro assays demonstrated that the substrate was oxidized to the fluorophore by horseradish peroxidase in the presence of hydrogen peroxide. In vivo uptake and oxidation of the substrate by Amoeba proteus was characterized by the initial appearance of fluorescent phagocytic vacuoles with subsequent localization in vesicular organelles the size and shape of protozoan mitochondria. Similar staining patterns occurred in cells incubated with substrate, oxidized rosamine or rhodamine 123, a known mitochondrial stain.