Synthesis and hydrolysis by cysteine and serine proteases of short internally quenched fluorogenic peptides

Synthesis and hydrolysis by cysteine and serine proteases of short internally quenched fluorogenic peptides

Autor Melo, R. L. Google Scholar
Alves, L. C. Google Scholar
Del Nery, E. Google Scholar
Juliano, L. Google Scholar
Juliano, M. A. Google Scholar
Instituição Universidade Federal de São Paulo (UNIFESP)
Resumo 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.
Assunto cathepsin B
cathepsin L
Idioma Inglês
Data 2001-06-01
Publicado em Analytical Biochemistry. San Diego: Academic Press Inc, v. 293, n. 1, p. 71-77, 2001.
ISSN 0003-2697 (Sherpa/Romeo, fator de impacto)
Editor Academic Press Inc
Extensão 71-77
Direito de acesso Acesso restrito
Tipo Artigo
Web of Science WOS:000169238800011

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