Interaction of heparin with internally quenched fluorogenic peptides derived from heparin-binding consensus sequences, kallistatin and anti-thrombin III

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dc.contributor.author Pimenta, Daniel C.
dc.contributor.author Nantes, Iseli L.
dc.contributor.author Souza, Eduardo S. de
dc.contributor.author Le Bonniec, Bernard
dc.contributor.author Ito, Amando S.
dc.contributor.author Tersariol, Ivarne Luis dos Santos [UNIFESP]
dc.contributor.author Oliveira, Vitor
dc.contributor.author Juliano, Maria Aparecida [UNIFESP]
dc.contributor.author Juliano, Luiz [UNIFESP]
dc.date.accessioned 2016-01-24T12:33:29Z
dc.date.available 2016-01-24T12:33:29Z
dc.date.issued 2002-09-01
dc.identifier http://dx.doi.org/10.1042/BJ20020023
dc.identifier.citation Biochemical Journal. London: Portland Press, v. 366, p. 435-446, 2002.
dc.identifier.issn 0264-6021
dc.identifier.uri http://repositorio.unifesp.br/handle/11600/26957
dc.description.abstract Internally quenched fluorogenic (IQF) peptides bearing the fluorescence donor/acceptor pair o-aminobenzoic acid (Abz)/N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) at N- and C-terminal ends were synthesized containing heparin-binding sites from the human serpins kallistatin and antithrombin, as well as consensus heparin-binding sequences (Cardin clusters). the dissociation constant (K-d), as well as the stoichiometry for the heparin-peptide complexes, was determined directly by measuring the decrease in fluorescence of the peptide solution. Experimental procedures were as sensitive as those used to follow the fluorescence change of tryptophan in heparin-binding proteins. the conformation of the peptides and the heparin-peptide complexes were obtained from measurements of time-resolved fluorescence decay and CD spectra. Kallistatin (Arg(300)-Pro(319))-derived peptide (HC2) and one derived from antithrombin III helix D [(AT3D), corresponding to Ser(112)-Lys(139)], which are the heparin-binding sites in these serpins, showed significant affinity for 4500 Da heparin, for which K-d values were 17 nM and 100 nM respectively. the CD spectra of the heparin-HC2 peptide complex did not show any significant alpha-helix content, different from the situation with peptide AT3D, for which complex-formation with heparin resulted in 24% alpha-helix content. the end-to-end distance distribution and the time-resolved fluorescence-decay measurements agree with the CD spectra and K-d values. the synthetic alpha-methyl glycoside pentasaccharide AGA*IA(M) (where A represents N,6-O-sulphated alpha-D-glucosamine; G, beta-D-glucuronic acid; A*, N,3, 6-O-sulphated alpha-D-glucosamine; 1, 2-O-sulphated alpha-L-iduronic acid; and A(M), alpha-methyl glycoside of A) also binds to AT3D and other consensus heparin-binding sequences, although with lower affinity. the interaction of IQF peptides with 4500 Da heparin was displaced by protamine. in conclusion, IQF peptides containing Abz/EDDnp as the donor/acceptor fluorescence pair are very promising tools for structure-activity relationship studies on heparin-peptide complexes, as well as for the development of new peptides as heparin reversal-effect compounds. en
dc.format.extent 435-446
dc.language.iso eng
dc.publisher Portland Press
dc.relation.ispartof Biochemical Journal
dc.rights Acesso aberto
dc.subject binding assay en
dc.subject glycosaminoglycan en
dc.subject kallikrein en
dc.subject time-resolved fluorescence en
dc.title Interaction of heparin with internally quenched fluorogenic peptides derived from heparin-binding consensus sequences, kallistatin and anti-thrombin III en
dc.type Artigo
dc.contributor.institution Universidade Federal de São Paulo (UNIFESP)
dc.contributor.institution CEPID
dc.contributor.institution UMC
dc.contributor.institution Universidade de São Paulo (USP)
dc.contributor.institution Univ Paris 05
dc.description.affiliation Universidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04044020 São Paulo, Brazil
dc.description.affiliation CEPID, CAT, Ctr Toxinol Aplicada, BR-05503900 São Paulo, Brazil
dc.description.affiliation UMC, CIIB, BR-08780911 Mogi Das Cruzes, SP, Brazil
dc.description.affiliation Univ São Paulo, FFCLRP, Dept Fis & Matemat, BR-14040901 Ribeirao Preto, SP, Brazil
dc.description.affiliation Univ Paris 05, Fac Pharm, INSERM, U428, F-75270 Paris 06, France
dc.description.affiliationUnifesp Universidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04044020 São Paulo, Brazil
dc.identifier.doi 10.1042/BJ20020023
dc.description.source Web of Science
dc.identifier.wos WOS:000177975500007



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