title: Synthesis and hydrolysis by cysteine and serine proteases of short internally quenched fluorogenic peptides.
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. All Abz-F-R-X-EDDnp synthesized peptides (for X = Phe, Leu, Ile, Ala, Pro, Gln, 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-R-A-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)). All Abz-F-R-X-EDDnp peptides were resistant to hydrolysis by thrombin and activated factor X.
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio 100, 04044-020 São Paulo, Brazil.
authors.forename: R L
authors.initials: RL
authors.lastname: Melo
authors.forename: L C
authors.initials: LC
authors.lastname: Alves
authors.forename: E
authors.initials: E
authors.lastname: Del Nery
authors.forename: L
authors.initials: L
authors.lastname: Juliano
authors.forename: M A
authors.initials: MA
authors.lastname: Juliano
mesh_terms: D002401:Cathepsin B / Q000378:metabolism*; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism*; D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D005170:Factor X / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007610:Kallikreins / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D010206:Papain / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis*; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D010446:Peptide Fragments; D005170:Factor X; D002403:Cathepsins; D010450:Endopeptidases; D007610:Kallikreins; D014357:Trypsin; D013917:Thrombin; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; D010206:Papain