A new methodology for the detection of three model proteases using a multi-purpose peptide probe equipped with three selectively cleavable sites and four fluorophores was developed and studied. The probe was designed as a single-excitation, triple-emission system, allowing for the monitoring of characteristic real-time changes in the fluorescence emission responses of individual fluorophores during enzymatic cleavage. It was labelled with diethylaminocoumarin (DEAC), fluorescein (FL) and Rhodamine B (RhB), forming the DEAC → FL → RhB FRET-cascade, with estimated Förster distances of 3.08 ± 0.02 nm or 3.02 ± 0.02 nm for the DEAC → FL FRET-pair (depending on the DEAC labeling site) and 6.04 ± 0.05 nm for the FL → RhB FRET-pair. Although spectroscopic analyses indicate that photophysical processes other than FRET are involved in the probe, the huge changes in its fluorescence intensities (e.g., the fluorescence intensity of the probe for the excitation/emission wavelengths 421 nm/476 nm is 50-fold lower compared to that of the DEAC-only labeled construct, or it is 200-fold lower for 480 nm/521 nm compared to that of the FL-only labeled construct) enable the easy detection of protease activities. In a single-enzyme mode, trypsin, chymotrypsin, and thrombin can be determined according to the ratiometric graphical model at the lowest detectable concentrations of 0.0625 ng mL-1, 0.125 μg mL-1, and 0.0125 U mL-1, respectively. The prepared probe, in combination with selective inhibitors (Kunitz, trypsin inhibitor; chymostatin, chymotrypsin inhibitor; and dabigatran, thrombin inhibitor), was also successfully used for the simultaneous detection of individual proteases in their two-enzyme mixtures. Finally, the probe was studied as a tool for three-protease screening using two selected inhibitors. Unfortunately, a universal model capable of unambiguous confirmation of the three studied enzymes in various combinations of their quantities has not been found.

Department of Experimental Physics Faculty of Science Palacký University Olomouc 17 Listopadu 12 771 46 Olomouc Czech Republic

Department of Organic Chemistry Faculty of Science Palacký University Olomouc 17 Listopadu 12 771 46 Olomouc Czech Republic

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