Internally quenched fluorescent peptide libraries with randomized sequences designed to detect endopeptidases

Internally quenched fluorescent peptide libraries with randomized sequences designed to detect endopeptidases

Author Oliveira, Lilian C. G. Autor UNIFESP Google Scholar
Silva, Vinicius O. Autor UNIFESP Google Scholar
Okamoto, Debora N. Autor UNIFESP Google Scholar
Kondo, Marcia Y. Autor UNIFESP Google Scholar
Santos, Saara M. B. Autor UNIFESP Google Scholar
Hirata, Izaura Yoshico Autor UNIFESP Google Scholar
Vallim, Marcelo A. Autor UNIFESP Google Scholar
Pascon, Renata C. Autor UNIFESP Google Scholar
Gouvea, Iuri E. Autor UNIFESP Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Abstract Identification of synthetic peptide substrates for novel peptidases is an essential step for their study. With this purpose we synthesized fluorescence resonance energy transfer (FRET) peptide libraries Abz (or MCA)-GXXXXXQ-EDDnp and Abs (or MCA)-GXXZXXQ-EDDnp, where X consists of an equimolar mixture of all amino acids, the Z position is fixed with one of the proteinogenic amino acids (cysteine was excluded), Abz (ortho-aminobenzoic acid) or MCA ([7-amino-4-methyl]coumarin) is the fluorescence donor and Q-EDDnp (glutamine-[N-(2,4-dinitrophenyl)-ethylenediamine]) is the fluorescence acceptor. the peptide libraries MCA-GXXX down arrow XXQ-EDDnp and MCA-GXXZ down arrow XXQ-EDDnp were cleaved as indicated (1) by trypsin, chymotrypsin, cathepsin L, pepsin A, and Eqolisin as confirmed by Edman degradation of the products derived from the digestion of these libraries. the best hydrolyzed Abz-GXXZXXQ-EDDnp sublibraries by these proteases, including Dengue 2 virus NS2B-NS3 protease, contained amino acids at the Z position that are reported to be well accepted by their S-1 subsite. the pH profiles of the hydrolytic activities of these canonical proteases on the libraries were similar to those reported for typical substrates. the FRET peptide libraries provide an efficient and simple approach for detecting nanomolar concentrations of endopeptidases and are useful for initial specificity characterization as performed for two proteases secreted by a Bacillus subtilis. (C) 2011 Elsevier Inc. All rights reserved.
Keywords Protease
Peptide libraries
Combinatorial libraries
Energy-transfer peptides
Substrate specificity
Trypsin
Chymotrypsin
Pepsin
Eqolisin
Cathepsin
Dengue
Language English
Sponsor Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Instituto Nacional de Fluidos Complexos (INCT-FCx)
Date 2012-02-01
Published in Analytical Biochemistry. San Diego: Academic Press Inc Elsevier Science, v. 421, n. 1, p. 299-307, 2012.
ISSN 0003-2697 (Sherpa/Romeo, impact factor)
Publisher Elsevier B.V.
Extent 299-307
Origin http://dx.doi.org/10.1016/j.ab.2011.10.025
Access rights Closed access
Type Article
Web of Science ID WOS:000299491300041
URI http://repositorio.unifesp.br/handle/11600/34577

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