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 (Kd), 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 (Arg300–Pro319)-derived peptide (HC2) and one derived from antithrombin III helix D [(AT3D), corresponding to Ser112–Lys139], which are the heparin-binding sites in these serpins, showed significant affinity for 4500Da heparin, for which Kd values were 17nM and 100nM respectively. The CD spectra of the heparin–HC2 peptide complex did not show any significant α-helix content, different from the situation with peptide AT3D, for which complex-formation with heparin resulted in 24% α-helix content. The end-to-end distance distribution and the time-resolved fluorescence-decay measurements agree with the CD spectra and Kd values. The synthetic α-methyl glycoside pentasaccharide AGA∗IAM (where A represents N,6-O-sulphated α-d-glucosamine; G, β-d-glucuronic acid; A∗, N,3,6-O-sulphated α-d-glucosamine; I, 2-O-sulphated α-l-iduronic acid; and AM, α-methyl glycoside of A) also binds to AT3D and other consensus heparin-binding sequences, although with lower affinity. The interaction of IQF peptides with 4500Da 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.
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September 2002
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Research Article|
September 01 2002
Interaction of heparin with internally quenched fluorogenic peptides derived from heparin-binding consensus sequences, kallistatin and anti-thrombin III
Daniel C. PIMENTA;
Daniel C. PIMENTA
∗Centro de Toxinologia Aplicada, CAT/CEPID, Av. Vital Brasil, 1500, São Paulo SP-05503-900, Brazil
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Iseli L. NANTES;
Iseli L. NANTES
†Centro Interdisciplinar de Investigação Bioquímica (CIIB), UMC, Avenida Cândido Xavier de Almeida Souza, 200, Mogi das Cruzes-SP, 08780-911, Brazil
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Eduardo S. de SOUZA;
Eduardo S. de SOUZA
‡Departamento de Física e Matemática, FFCLRP, Universidade de São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, SP 14040-901, Brazil,
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Bernard Le BONNIEC;
Bernard Le BONNIEC
§INSERM, U428, Université Paris V, Faculté de Pharmacie, 4 Avenue de l'Observatoire, 75270 Paris, cedex 06, France,
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Amando S. ITO;
Amando S. ITO
‡Departamento de Física e Matemática, FFCLRP, Universidade de São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, SP 14040-901, Brazil,
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Ivarne L.S. TERSARIOL;
Ivarne L.S. TERSARIOL
†Centro Interdisciplinar de Investigação Bioquímica (CIIB), UMC, Avenida Cândido Xavier de Almeida Souza, 200, Mogi das Cruzes-SP, 08780-911, Brazil
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Vitor OLIVEIRA;
Vitor OLIVEIRA
∥Universidade Federal de São Paulo, Escola Paulista de Medicina, Departmento de Biofísica, Rua Três de Maio, 100, São Paulo 04044-020, Brazil
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Maria A. JULIANO;
Maria A. JULIANO
∥Universidade Federal de São Paulo, Escola Paulista de Medicina, Departmento de Biofísica, Rua Três de Maio, 100, São Paulo 04044-020, Brazil
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Luiz JULIANO
Luiz JULIANO
1
∥Universidade Federal de São Paulo, Escola Paulista de Medicina, Departmento de Biofísica, Rua Três de Maio, 100, São Paulo 04044-020, Brazil
1To whom correspondence should be addressed (e-mail juliano.biof@epm.br).
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Publisher: Portland Press Ltd
Received:
January 04 2002
Revision Received:
May 02 2002
Accepted:
May 08 2002
Accepted Manuscript online:
May 08 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 366 (2): 435–446.
Article history
Received:
January 04 2002
Revision Received:
May 02 2002
Accepted:
May 08 2002
Accepted Manuscript online:
May 08 2002
Citation
Daniel C. PIMENTA, Iseli L. NANTES, Eduardo S. de SOUZA, Bernard Le BONNIEC, Amando S. ITO, Ivarne L.S. TERSARIOL, Vitor OLIVEIRA, Maria A. JULIANO, Luiz JULIANO; Interaction of heparin with internally quenched fluorogenic peptides derived from heparin-binding consensus sequences, kallistatin and anti-thrombin III. Biochem J 1 September 2002; 366 (2): 435–446. doi: https://doi.org/10.1042/bj20020023
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