Proteinase assay by capillary electrophoresis employing fluorescence-quenched protein-dye conjugates

Abstract

Determination of proteinases--enzymes that catalyze the hydrolysis of peptide bonds--is often difficult due to the presence of interferences in complex biological media and limited sample size. Capillary electrophoresis (CE), with laser-induced fluorescence (LIF) can serve as a useful tool for such determinations. LIF detection offers the advantages of increased sensitivity and increased selectivity. However, direct LIF detection requires the proteinase analyte to be fluorescently derivatized prior to analysis. A viable alternative is offered by the present work, in which protein substrates are first labeled with BODIPY dye, a relatively pH-insensitive, high-fluorescence quantum yield dye. Upon binding of some 4-10 molecules of dye to a single protein, the dye is effectively fluorescence-quenched. Digestion of the BODIPY--labeled and quenched protein by an unlabeled enzyme yields smaller peptide fragments in which the fluorescence of associated BODIPY tags is restored. We will present how the fragmentation pattern of BODIPY-labeled casein changes as a function of incubation time with trypsin, as well as the effect of varying concentrations of trypsin on the BODIPY-casein digest.

In situ detection of protein-hydrolysing microorganisms in activated sludge

“…BODIPY dye‐labeled proteins have previously mainly been used to study the protease activity of purified enzymes (e.g. Welder et al , 2002), and only recently have they been applied also to study protease activity directly in a bacterial culture (Yoshioka et al , 2003). Our study has shown that BODIPY FL proteins can also be used successfully to label and identify PHOs in combination with FISH in activated sludge and thus gain more knowledge regarding the organisms involved in hydrolysis and consumption of proteins in complex microbial communities.…”

“…The quenched fluorescence is released in the presence of proteases (Jones et al, 1997). Based on this, BODIPY dye-labeled substrates [casein and bovine serum albumin (BSA)] have been used to measure protease activity of pure enzymes (Welder et al, 2002) and recently also directly in pure cultures of the Gram-negative Porphyromonas gingivalis (Yoshioka et al, 2003). However, besides a precipitation of fluorescent BODIPY dye on the surface of P. gingivalis some intracellular uptake of fluorescent hydrolysates has also been reported, causing the bacterial cells to fluoresce (Yoshioka et al, 2003).…”

A capillary electrophoretic assay for ribonuclease H activity

“…Capillary electrophoretic techniques have been previously applied to enzymatic assays of proteases [5] and kinases [6,7]. With currently available automation it is possible to substantially increase throughput in this type of assay by using a 96-capillary-array electrophoretic instrument [7].…”

In situ identification of keratin-hydrolyzing organisms in swine manure inoculated anaerobic digesters

“…BODIPY FL casein has mainly been used to study protease activity of purified enzymes (e.g. Welder et al, 2002). Only quite recently has it been applied to study protease activity in situ in bacterial cells (Yoshioka et al, 2003) and in uncultured bacterial populations in communities of activated sludge wastewater treatment plants (Xia et al, 2007;Xia et al, 2008).…”