Dynamics of digestive proteolytic system during blood feeding of the hard tick Ixodes ricinus

Parasites & Vectors

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-AMC^a (5 μM)	-	360/465	CA-074^c
IrCL	4.0	Z- Phe-Arg-AMC^a (5 μM)	CA-074^c (2.5 μM)	360/465	n.d.^e
IrCC	5.5	Gly-Arg-AMC^a (40 μM)	-	360/465	Ala-Hph-VS-Ph^f
IrAE	5.5	Z-Ala-Ala-Asn-AMC^a (10 μM)	CA-074^c (2.5 μM)	360/465	Aza-N-11a^g
IrCD	4.0	Abz-Lys-Pro-Ala-Glu-Phe-Nph-Ala-Leu^b (32 μM)	E-64^d (5 μM)	330/425	pepstatin^h

^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].

ISSN: 1756-3305