Table 1 Kinetic parameters of four internally quenched substrates with seven proteases of various catalytic types.

Enzyme and substrate	Substrate containing ACC/Lys(DNP)			Substrate containing MCA/Lys(DNP)
Enzyme and substrate	k_cat, s⁻¹	K_M, μM	k_cat/K_M, M⁻¹s⁻¹	k_cat, s⁻¹	K_M, μM	k_cat/K_M, M⁻¹s⁻¹
Caspase-3 (1.0/3.0 nM)
Donor-GDEVD*GVK(DNP)D-NH₂	7.52	6.74	1 116 000	11.24	4.67	2 359 000
Caspase-7 (10/30 nM)
Donor-GDEVD*GVK(DNP)D-NH₂	0.47	8.36	56 800	0.68	6.17	106 000
Caspase-8 (20/50 nM)
Donor-GDEVD*GVK(DNP)D-NH₂	0.32	11.0	29 400	0.32	11.7	26 300
Legumain (7.3/24.5 nM)
Donor-GPTN*KVK(DNP)R-NH₂	1.35	9.13	149 000	1.09	8.11	139 000
Elastase (0.34/1.4 nM)
Donor-GAEPV*SLK(DNP)L-NH₂	2.44	1.22	2 006 000	2.58	2.27	1 147 219
MMP2 (1.25/3.8 nM)
Donor-GPLG*LK(DNP)AR-NH₂	4.26	15.4	278 800	6.24	35.1	179 900
MMP9 (2.4/7.2 nM)
Donor-GPLG*LK(DNP)AR-NH₂	0.69	7.81	89 200	0.958	11.7	82 400

Standard deviations were obtained using at least three independent experiments for each measurement and were less than 10%. For each fluorophore, different enzyme concentrations were used (for example, for caspase-3, 1 nM was used for ACC-containing substrates, and 3 nM was used for MCA-containing substrates).

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