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

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.

Capillary electrophoresis with laser-induced fluorescence detection of indigo carmine and indigo carmine-labeled proteins.

Indigo carmine blue is a water-soluble, light-sensitive, anionic dye most widely applied in microscopic staining techniques. It is virtually nonfluorescent in its oxidized state in aqueous solution, but when reduced under alkaline conditions, it becomes fluorescent, with absorption and emission maxima at 436 and 528 nm, respectively. It is demonstrated that the fluorescent character of the reduced form of the dye can be exploited as a label for the determination of cationic proteins by capillary electrophoresis with laser-induced fluorescence detection. Model proteins trypsinogen and cytochrome c are noncovalently labeled with indigo carmine; the bound species are reduced, rendering the indigo carmine fluorescent; and capillary electrophoresis with laser-induced fluorescence detection is used for subsequent analysis. Various buffer systems and buffer additives were examined and optimized, and a suitable pH range for optimal fluorescence intensity and protein-dye interaction was established. Fluorescence quenching of the reduced dye when bound to protein was observed in all buffer systems.