Navegando por Palavras-chave "protease fluorescent substrate"

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    End-to-end distance distribution in bradykinin observed by Forster resonance energy transfer
    (Elsevier B.V., 2000-04-06) Souza, E. S. de; Hirata, I. Y.; Juliano, L.; Ito, A. S.; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)
    Forster resonance energy transfer (FRET) was used to study the conformational dynamics of bradykinin related peptides. the fluorescent probe aminobenzoic acid (Abz) bound to the amino terminal of bradykinin maintained its fluorescence characteristics, like high quantum yield and excited state decay dominated by a lifetime of 8.3 ns. the binding of the acceptor group N-[2,4-dinitrophenyl]-ethylenediamine (EDDnp) to the carboxy terminal of Abz labeled bradykinin resulted in a drastic decrease of the fluorescence intensity and in a fastening of the excited state decay. the change of the decay kinetics to an heterogeneous process, precludes the use of energy transfer models based on a single fixed distance between donor and acceptor. the computational package CONTIN was employed to the analysis of time-resolved fluorescence data, allowing the recovery of a distance distribution between donor and acceptor corresponding to the end-to-end distance of the labeled peptide. the distance distribution reflects the occurrence of distinct conformations for the peptide, that coexist in equilibrium during the fluorescence lifetime. We observed three distance populations for bradykinin in water, that merged to two populations when the solvent was trifluoroethanol (TFE). the results were consistent with those obtained from circular dichroism spectroscopy, that showed structural flexibility in water and the presence of more defined secondary structure in TFE. We also studied several peptides related to bradykinin, and the results emphasized the formation of turns involving the proline residues and the decrease of conformational flexibility induced by using TFE as the solvent. (C) 2000 Elsevier Science B.V. All rights reserved.
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    Reduction of ortho-aminobenzoyl-proline fluorescence and formation of pyrrolobenzodiazepine-5,11-dione
    (Kluwer Academic Publ, 1998-01-01) Hirata, Izaura Yoshico [UNIFESP]; Cezari, Maria Helena Sedenho [UNIFESP]; Boschcov, Paulo [UNIFESP]; Garratt, Richard Charles; Oliva, Glaucius; Ito, Amando Siuiti; Spisni, Alberto; Franzoni, Lorella; Juliano, Luiz [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); Univ Parma
    The ortho-aminobenzoic acid (Abz) group is widely employed as a fluorescent marker for peptides used as substrates for the study of proteolytic enzyme activity. in fact, a direct correlation has been observed between fluorescence intensity and enzyme activity. An unusual behavior of the fluorescence properties of this group, which would lead to erroneous evaluation of the enzyme activity, was observed when it is bound directly to proline. Here we report a systematic NMR, fluorescence and X-ray diffraction study of the compounds obtained from Boc-Abz-Pro-NH2, Boc-Abz-Pro-OH, as well as from various other Boc-Abz-Pro-X derivatives, after treatment with HCl or TFA under anhydrous conditions. We verified that, as recently reported, even under these synthetic conditions, deprotection of Boc-Abz-Pro-NH2 or Boc-Abz-Pro-OH leads to the formation of the same product: pyrrolobenzodiazepine-5,11-dione. However, the formation of this compound was not detected with Abz-Pro-N(CH3)(2), Abz-Pro-Leu-Gly-NH2 or Abz-pyrrolidine. for all these compounds we observed an unusual behavior for the fluorescence quantum yield of Abz that can be explained as the consequence of a non-radiative deactivation process produced, specifically, by the amidation of the Abz carboxyl group with proline or a similar secondary amine such as pyrrolidine. in conclusion, these results indicate that Abz cannot be used as an internal fluorescence marker for proteolytic enzyme activity when bound directly to proline.