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Internally quenched fluorogenic protease substrates: Solid-phase synthesis and fluorescence spectroscopy of peptides containing ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs
Authors:Izaura Yoshico Hirata   Maria Helena Sedenho Cezari   Clovis Ryuichi Nakaie   Paulo Boschcov   Amando Siuiti Ito   Maria Aparecida Juliano  Luiz Juliano
Affiliation:(1) Department of Biophysics, Escola Paulista de Medicina, Rua Tres de Maio 100, 04044-020 São Paulo, Brazil;(2) Institute of Physics, Universidade de São Paulo, 01498-970 São Paulo, Brazil
Abstract:
Summary A general procedure, using the commonly employed solid-phase peptide synthesis methodology for obtaining internally quenched fluorogenic peptides with ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs, is presented. The essential feature of this procedure is the synthesis of an Nagr-Boc or-Fmoc derivative of glutamic acid with the agr-carboxyl group bound to N-(2,4-dinitrophenyl)-ethylenediamine (EDDnp), which provides the quencher moiety attached to the C-terminus of the substrate. The fluorescent donor group, ortho-aminobenzoic acid (Abz), is incorporated into the resin-bound peptide in the last coupling cycle. Depending on the resin type used, Abz-peptidyl-Gln-EDDnp or Abz-peptidyl-Glu-EDDnp is obtained. Using the procedure described above, substrates for human renin and tissue kallikreins were synthesised. Spectrofluorimetric measurements of Abz bound to the agr-amino group of proline showed that strong quenching of Abz fluorescence occurs in the absence of any acceptor group.
Keywords:Protease fluorescent substrate  Renin  Tissue kallikrein  Peptide synthesis  Ortho-aminobenzoyl-proline
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