Table 2.
Enzymes, substrates and buffer used to verify substrate hydrolysis by serino proteases and inhibitory activity.
| Enzyme | Substrate | Buffer |
|---|---|---|
| trypsin (20 μL, 0.41 μM) * NPGB |
Bz-Arg-pNan (25 μL, 10 mM) | Tris/HCl 0.1 M, pH 8.0, CaCl2 0.02% (v/v) |
| HNE (20 μL, 0.21 μM) (* α1-anti-trypsin) | MeO-Suc-Ala-Ala-Pro-Val-pNan (25 μL, 1.1 mM) |
Tris/HCl 0.1 M, pH 7.0, NaCl 0.5 M |
| PKa (20 μL, 0.84 μM) (* EcTI) | H-D-Pro-Phe-Arg-pNan (25 μL, 5 mM) |
Tris/HCl 0.05 M, pH 8.0 |
| PoPK (30 μL, 0.16 nM) (* aprotinin) | H-D-Pro-Phe-Arg-AMC (30 μL, 5 mM) |
Tris/HCl 0.1 M, pH 9.0, plus albumin 0.1% (v/v) |
| chymotrypsin (40 μL, 0.88 µM) (* EcTI) | Suc-Phe-pNan (20 μL, 20 mM) | Tris/HCl 0.1 M, pH 8.0, CaCl2 0.02% (v/v) |
| plasmin (25 μL, 0.028 μM) (* BvTI) | H-D-Val-Leu-Lys-pNan (20 μL, 9 mM) |
Tris/HCl 0.1 M, pH 7.4, NaCl 0.2 M |
| thrombin (10 μL, 0.267 μM) (* rhodinin) | H-D-Phe-L-Pip-L-Arg-pNan (20 μL, 2 mM) |
Tris/HCl 0.05 M, pH 8.0 |
| factor Xa (30 μL, 0.467 μM) (* BuXI) | Boc-Ile-Glu-Gly-Arg-AMC (60 μL, 6 mM) |
Tris/HCl 0.05 M, pH 8.0 |
| cathepsin G (30 μL, 0.25 μM) (* α1-anti-trypsin) | N-Suc-Ala-Ala-Pro-Phe-pNan (25 μL, 1 mM) |
Tris/HCl 0.05 M, pH 7.0, NaCl 0.5 M |
* inhibitor used for enzyme titration. HNE: human neutrophil elastase; PKa: human plasma kallikrein; PoPK: porcine pancreatic kallikrein.