Table 1.
Assay conditions for the fluorimetric measurements of digestive peptidases activities and stoichiometric active-site titration
| Enzyme | pH | Substrate (final concentration) |
Shielding inhibitor (final concentration) |
Excitation/Emission [nm] | Active-site titration inhibitor |
|---|---|---|---|---|---|
| IrCB | 5.5 | Z-Arg-Arg-AMCa (5 μM) |
- | 360/465 | CA-074c |
| IrCL | 4.0 | Z- Phe-Arg-AMCa (5 μM) |
CA-074c (2.5 μM) | 360/465 | n.d.e |
| IrCC | 5.5 | Gly-Arg-AMCa (40 μM) |
- | 360/465 | Ala-Hph-VS-Phf |
| IrAE | 5.5 | Z-Ala-Ala-Asn-AMCa (10 μM) |
CA-074c (2.5 μM) | 360/465 | Aza-N-11ag |
| IrCD | 4.0 | Abz-Lys-Pro-Ala-Glu-Phe-Nph-Ala-Leub (32 μM) |
E-64d (5 μM) | 330/425 | pepstatinh |
a The fluorogenic AMC-substrates (AMC, 7-amino-4-methylcoumarin) were from Bachem
b Fluorescence resonance energy transfer (FRET) substrate [38]
c Epoxysuccinyl-based inhibitor of cathepsin B [39]
d Epoxysuccinyl-based inhibitor of papain-type cysteine peptidases (cathepsins C, B and L) [34]
e n.d. - not determined due to a lack of an appropriate titrant
f Vinyl sulfone-based inhibitor of cathepsin C [40]
g Aza-peptide Michael acceptor-based inhibitor of asparaginyl endopeptidases [41]
h Statine-based inhibitor of aspartic peptidases [35].