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

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

Autor Pimenta, Daniel C. Google Scholar
Nantes, Iseli L. Google Scholar
Souza, Eduardo S. de Google Scholar
Le Bonniec, Bernard Google Scholar
Ito, Amando S. Google Scholar
Tersariol, Ivarne Luis dos Santos Autor UNIFESP Google Scholar
Oliveira, Vitor Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Instituição Universidade Federal de São Paulo (UNIFESP)
CEPID
UMC
Universidade de São Paulo (USP)
Univ Paris 05
Resumo 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.
Palavra-chave binding assay
glycosaminoglycan
kallikrein
time-resolved fluorescence
Idioma Inglês
Data de publicação 2002-09-01
Publicado em Biochemical Journal. London: Portland Press, v. 366, p. 435-446, 2002.
ISSN 0264-6021 (Sherpa/Romeo, fator de impacto)
Publicador Portland Press
Extensão 435-446
Fonte http://dx.doi.org/10.1042/BJ20020023
Direito de acesso Acesso aberto Open Access
Tipo Artigo
Web of Science WOS:000177975500007
Endereço permanente http://repositorio.unifesp.br/handle/11600/26957

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