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A new proteinase 3 substrate with improved selectivity over human neutrophil elastase
- Popow-Stellmaszyk, J., Wysocka, M., Lesner, A., Korkmaz, B., Rolka, K.
- Analytical biochemistry 2013 v.442 no.1 pp. 75-82
- elastase, fluorescence, humans, neutrophils
- We report the synthesis and enzymatic studies on a new proteinase 3 intermolecular quenched substrate with enhanced selectivity over neutrophil elastase. Using combinatorial chemistry methods, we were able to synthesize the hexapeptide library with the general formula ABZ-Tyr-Tyr-Abu-X₁′-X₂′-X₃′-Tyr(3-NO₂)-NH₂ using the mix and split method. The iterative deconvolution of such a library allowed us to obtain the sequence ABZ-Tyr-Tyr-Abu-Asn-Glu-Pro-Tyr(3-NO₂)-NH₂ with a high specificity constant (kcₐₜ/KM=1534×10³M⁻¹s⁻¹) and superior selectivity over neutrophil elastase and other neutrophil-derived serine proteases. Moreover, using the obtained substrate, we were able to detect a picomolar concentration of proteinase 3 (PR3). Incubation of the above-mentioned substrate with neutrophil lysate resulted in a strong fluorescent signal that was significantly reduced in the presence of a PR3 selective inhibitor.