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.authorPimenta, Daniel C.
dc.contributor.authorNantes, Iseli L.
dc.contributor.authorSouza, Eduardo S. de
dc.contributor.authorLe Bonniec, Bernard
dc.contributor.authorIto, Amando S.
dc.contributor.authorTersariol, Ivarne Luis dos Santos [UNIFESP]
dc.contributor.authorOliveira, Vitor
dc.contributor.authorJuliano, Maria Aparecida [UNIFESP]
dc.contributor.authorJuliano, Luiz [UNIFESP]
dc.contributor.institutionUniversidade Federal de São Paulo (UNIFESP)
dc.contributor.institutionCEPID
dc.contributor.institutionUMC
dc.contributor.institutionUniversidade de São Paulo (USP)
dc.contributor.institutionUniv Paris 05
dc.date.accessioned2016-01-24T12:33:29Z
dc.date.available2016-01-24T12:33:29Z
dc.date.issued2002-09-01
dc.description.abstractInternally 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.description.affiliationUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04044020 São Paulo, Brazil
dc.description.affiliationCEPID, CAT, Ctr Toxinol Aplicada, BR-05503900 São Paulo, Brazil
dc.description.affiliationUMC, CIIB, BR-08780911 Mogi Das Cruzes, SP, Brazil
dc.description.affiliationUniv São Paulo, FFCLRP, Dept Fis & Matemat, BR-14040901 Ribeirao Preto, SP, Brazil
dc.description.affiliationUniv Paris 05, Fac Pharm, INSERM, U428, F-75270 Paris 06, France
dc.description.affiliationUnifespUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04044020 São Paulo, Brazil
dc.description.sourceWeb of Science
dc.format.extent435-446
dc.identifierhttp://dx.doi.org/10.1042/BJ20020023
dc.identifier.citationBiochemical Journal. London: Portland Press, v. 366, p. 435-446, 2002.
dc.identifier.doi10.1042/BJ20020023
dc.identifier.issn0264-6021
dc.identifier.urihttp://repositorio.unifesp.br/handle/11600/26957
dc.identifier.wosWOS:000177975500007
dc.language.isoeng
dc.publisherPortland Press
dc.relation.ispartofBiochemical Journal
dc.rightsinfo:eu-repo/semantics/openAccess
dc.subjectbinding assayen
dc.subjectglycosaminoglycanen
dc.subjectkallikreinen
dc.subjecttime-resolved fluorescenceen
dc.titleInteraction of heparin with internally quenched fluorogenic peptides derived from heparin-binding consensus sequences, kallistatin and anti-thrombin IIIen
dc.typeinfo:eu-repo/semantics/article
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