Abstract:Fluorogenic probes that are activated by an enzymatic transformation are ideally suited for profiling enzyme activities in biological systems. Here, we describe two fluorogenic enzyme probes, 3-O-methylfluorescein-sulfate and resorufin-sulfate, that can be used to detect sulfatases in mycobacterial lysates. Both probes were validated with a set of commercial sulfatases and used to reveal species specific sulfatase banding patterns in a gel-resolved assay of mycobacterial lysates. The fluorogenic probes describ… Show more
“…[10] Furthermore, we recently applied fluorogenic probes to examine mycobacterial sulfatases in a protein gel-based assay format. [11, 12] We found that although most mycobacterial species, including M. tuberculosis , could hydrolyze sulfated fluorophores, the pattern of sulfatase activity varied from one species to another.…”
mentioning
confidence: 98%
“…[16–22] Fluorogenic enzyme probes could directly detect enzyme activities in intact organisms [9, 10] or in protein gel-resolved lysates without the need for isolated enzyme. [11, 12, 23] …”
mentioning
confidence: 99%
“…[16][17][18][19][20][21][22] Fluorogenic enzyme probes could directly detect enzyme activities in intact organisms [9,10] or in protein gel-resolved lysates without the need for isolated enzyme. [11,12,23] The simplest fluorogenic esterase probes are fluorophores masked by acetate esters (e.g., fluorescein diacetate, FDA), but these can suffer from high background fluorescence due to significant levels of spontaneous hydrolysis. [24][25][26] Thus, researchers have developed a variety of masking strategies to create more stable substrates.…”
Fluorogenic enzyme probes go from a dark to a bright state following hydrolysis and can provide a sensitive, real-time readout of enzyme activity. They are useful for examining enzymatic activity in bacteria, including the human pathogen Mycobacterium tuberculosis. Herein, we describe two fluorogenic esterase probes derived from the far-red fluorophore 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). These probes offer enhanced optical properties compared to existing esterase probes because the hydrolysis product, DDAO, excites above 600 nm while retaining a good quantum yield (ϕ=0.40). We validated both probes with a panel of commercially available enzymes alongside known resorufin- and fluorescein-derived esterase substrates. Furthermore, we used these probes to reveal esterase activity in protein gel-resolved mycobacterial lysates. These probes represent new tools for esterase detection and characterization and should find use in a variety of applications.
“…[10] Furthermore, we recently applied fluorogenic probes to examine mycobacterial sulfatases in a protein gel-based assay format. [11, 12] We found that although most mycobacterial species, including M. tuberculosis , could hydrolyze sulfated fluorophores, the pattern of sulfatase activity varied from one species to another.…”
mentioning
confidence: 98%
“…[16–22] Fluorogenic enzyme probes could directly detect enzyme activities in intact organisms [9, 10] or in protein gel-resolved lysates without the need for isolated enzyme. [11, 12, 23] …”
mentioning
confidence: 99%
“…[16][17][18][19][20][21][22] Fluorogenic enzyme probes could directly detect enzyme activities in intact organisms [9,10] or in protein gel-resolved lysates without the need for isolated enzyme. [11,12,23] The simplest fluorogenic esterase probes are fluorophores masked by acetate esters (e.g., fluorescein diacetate, FDA), but these can suffer from high background fluorescence due to significant levels of spontaneous hydrolysis. [24][25][26] Thus, researchers have developed a variety of masking strategies to create more stable substrates.…”
Fluorogenic enzyme probes go from a dark to a bright state following hydrolysis and can provide a sensitive, real-time readout of enzyme activity. They are useful for examining enzymatic activity in bacteria, including the human pathogen Mycobacterium tuberculosis. Herein, we describe two fluorogenic esterase probes derived from the far-red fluorophore 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). These probes offer enhanced optical properties compared to existing esterase probes because the hydrolysis product, DDAO, excites above 600 nm while retaining a good quantum yield (ϕ=0.40). We validated both probes with a panel of commercially available enzymes alongside known resorufin- and fluorescein-derived esterase substrates. Furthermore, we used these probes to reveal esterase activity in protein gel-resolved mycobacterial lysates. These probes represent new tools for esterase detection and characterization and should find use in a variety of applications.
A new probe for detecting sulfatase activity generated fluorescent N-methylisoindole when it was treated with sulfatase. Detailed synthetic procedures of reaction intermediates are described along with their spectral data. The Diels-Alder adduct of N-methylisoindole and N-phenylmaleimide was identified by LC–MS data. The fluorescence changes and kinetic values of the probe upon sulfatase treatment in the presence and absence of reducing agents were described. Inhibitory potency of EMATE was measured using the probe in the presence and absence of reducing agents. In addition, the fluorescence intensities of the probe upon sulfatase treatment were also monitored at pH 5 and 7 with and without reducing agents.
“…Activitybased probes have been designed to detect a number of bacterial proteins, such as sulfatases 85,86 , serine proteases 87 , the cell wall biosynthetic protein MurA 88 , glycoside hydrolases 89 , redox-sensitive proteins 90,91 and histidine kinases 92 ; however, most of these probes have been used only in gel-based analyses. One additional strategy that has been applied to bacterial imaging is the use of a probe that becomes fluorescent upon turnover by nitroreductase.…”
Fluorescence microscopy is an essential tool for the exploration of cell growth, division, transcription and translation in eukaryotes and prokaryotes alike. Despite the rapid development of techniques to study bacteria, the size of these organisms (1–10 μm) and their robust and largely impenetrable cell envelope present major challenges in imaging experiments. Fusion-based strategies, such as attachment of the protein of interest to a fluorescent protein or epitope tag, are by far the most common means for examining protein localization and expression in prokaryotes. While valuable, the use of genetically encoded tags can result in mislocalization or altered activity of the desired protein, does not provide a readout of the catalytic state of enzymes and cannot enable visualization of many other important cellular components, such as peptidoglycan, lipids, nucleic acids or glycans. Here, we highlight the use of biomolecule-specific small-molecule probes for imaging in bacteria.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
