| dc.description.abstract | We developed sensitive substrates for cysteine proteases and specific substrates for serine proteases based on short internally quenched fluorescent peptides, Abz-F-R-X-EDDnp, where Abz (ortho-aminobenzoic acid) is the fluorescent donor, EDDnp [N-(ethylenediamine) -2,4-dinitrophenyl amide] is the fluorescent quencher, and X are natural amino acids. This series of peptides is compared to the commercially available Z-F-R-MCA, where Abz and X replace carbobenzoxy (Z) and methyl-7-aminocoumarin amide (MCA), respectively; and EDDnp can be considered a P-2' residue. Whereas MCA is the fluorescent probe and cannot be modified, in the series Abz-F-R-X-EDDnp the amino acids X give the choice of matching the specificity of the S-1' enzyme subsite, increasing the substrate specificity for a particular protease. AU Abz-F-R-X-EDDnp synthesized peptides (for X = Phe, Leu, ne, Ala, Pro, Gin, Ser, Lys, and Arg) were assayed with papain, human cathepsin L and B, trypsin, human plasma, and tissue kallikrein. Abz-F-R-L-EDDnp was the best substrate for papain and Abz-F-R-R-EDDnp or Abz-F-RA-EDDnp was the more susceptible to cathepsin L. Abz-F-R-L-EDDnp was able to detect papain in the range of 1 to 15 pM. Human plasma kallikrein hydrolyzed Abz-F-R-R-EDDnp with significant efficiency (k(cat)/K-m = 1833 mM(-1) s(-1)) and tissue kallikrein was very selective, hydrolyzing only the peptides Abz-F-R-A-EDDnp (K-cat/K-m = 2852 mM(-1) s(-1)) and Abz-F-R-S-EDDnp (k(cat)/K-m = 4643 mM(-1) s(-1)). Ah Abz-F-R-X-EDDnp peptides were resistant to hydrolysis by thrombin and activated factor X. (C) 2001 Academic Press. | |
