Determination and reoxidation of the disulfide bridges of a squash-type trypsin inhibitor from Sechium edule seeds

Determination and reoxidation of the disulfide bridges of a squash-type trypsin inhibitor from Sechium edule seeds

Autor Faca, V. M. Google Scholar
Pereira, SR Google Scholar
Laure, M. J. Google Scholar
Greene, L. J. Google Scholar
Instituição Universidade Federal de São Paulo (UNIFESP)
Universidade de São Paulo (USP)
Resumo The determination of the disulfide pairings of SETI-II, a trypsin inhibitor isolated from Sechium edule, is described herein. the inhibitor contains 31 amino acid residues per mol, 6 of which are cysteine. Forty-five nmol (160 mug) of SETI-II was hydrolyzed with 20 mug thermolysin for 48 hr at 45degreesC, and peptides were separated by reverse phase high performance liquid chromatography (RP-HPLC). the major products were identified by amino acid composition, Edman degradation, and on the basis of the sequence of the inhibitor. the disulfide bridge pairings and (yields) are: Cys1-Cys4 (79%), Cys2-Cys5 (21%) and Cys3-Cys6 (43%). When the reduced inhibitor was reoxidized with glutathione reduced form (GSH)/glutathione oxidized form (GSSG) at pH 8.5 for 3 hr, full activity was recovered. These data show that disulfide bridge pairing and oxidation can be determined at nanomole levels and that sensitive and quantitative Edman degradation can eliminate the final time- and material-consuming step of disulfide determinations by eliminating the need to purify and cleave each peptide containing a disulfide bridge.
Assunto disulfide bridge determination
disulfide reoxidation
squash trypsin inhibitors
Sechium edule
thermolysin
Idioma Inglês
Data 2004-07-01
Publicado em Protein Journal. New York: Kluwer Academic/plenum Publ, v. 23, n. 5, p. 309-315, 2004.
ISSN 1572-3887 (Sherpa/Romeo, fator de impacto)
Editor Kluwer Academic/plenum Publ
Extensão 309-315
Fonte http://dx.doi.org/10.1023/B:JOPC.0000032650.40260.e2
Direito de acesso Acesso restrito
Tipo Artigo
Web of Science WOS:000223042600002
URI http://repositorio.unifesp.br/handle/11600/27827

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