RESUMO
The interaction of apramycin with copper at different pH values was investigated by potentiometric titrations and EPR, UV-vis and CD spectroscopic techniques. The Cu(II)-apramycin complex prevailing at pH 6.5 was further characterized by NMR spectroscopy. Metal-proton distances derived from paramagnetic relaxation enhancements were used as restraints in a conformational search procedure in order to define the structure of the complex. Longitudinal relaxation rates were measured with the IR-COSY pulse sequence, thus solving the problems due to signal overlap. At pH 6.5 apramycin binds copper(II) with a 2 : 1 stoichiometry, through the vicinal hydroxyl and deprotonated amino groups of ring III. Plasmid DNA electrophoresis showed that the Cu(II)-apramycin complex is more active than free Cu(II) in generating strand breakages. Interestingly, this complex in the presence of ascorbic acid damages DNA with a higher yield than in the presence of H(2)O(2).
Assuntos
Cobre/química , DNA/química , Nebramicina/análogos & derivados , Estresse Oxidativo/efeitos dos fármacos , Plasmídeos/química , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Dicroísmo Circular , Cobre/metabolismo , DNA/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Químicos , Nebramicina/química , Nebramicina/metabolismo , Oxidantes/farmacologia , TermodinâmicaRESUMO
The effect of copper(II) ions on the probabilities of existence of the four detectable conformers of the tetrapeptide Tyr-Pro-Phe-Pro (beta-casomorphin 4) in [2H6]DMSO was investigated by 1H NMR spectroscopy. Integration of the Phe-NH signals provided the relative populations in the free state as tt/tc/ct/cc=28:34:29:9 at 293 K (c=cis, t=trans). Copper(II) was shown to bind to all four isomers, yielding complexes with two different structures, depending on the conformation of Pro(2). The interpretation of paramagnetic relaxation rates of Pro(2)-Halpha signals provided the corresponding isomeric probabilities in the metal-bound state as 13:36:20:31. The observed stabilization of the conformation with the lowest probability of existence (cc) may be relevant for the biological role of copper and other metal ions.
Assuntos
Cobre/química , Endorfinas/química , Fragmentos de Peptídeos/química , Prolina/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica , Soluções , Estereoisomerismo , TermodinâmicaRESUMO
The NMR features of bradykinin were investigated in dimethylsulfoxide containing 1% water. The temperature dependence of chemical shifts and ROESY maps were monitored for the major species where all X-Pro bonds are trans. The occurrence of a head-to-tail ionic interaction and intramolecular hydrogen bonds stabilizing a pseudo cyclic arrangement was inferred, a beta turn at the C-terminus being the main feature of the secondary structure. Calcium was shown to bind to the peptide with a dissociation constant Kd = 2.8 + 0.2 mm. 2Pro and 3Pro carbonyls, as well as the 9Arg carboxyl, were assigned as the metal-binding sites. A molecular model of the 1 : 1 metal-complex was obtained. In light of conformational changes experienced by the peptide upon interaction with calcium, a role for the metal was hypothesized in the process of conformational selection from the free to the receptor-bound state of bradykinin.
Assuntos
Bradicinina/metabolismo , Cálcio/metabolismo , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Ligação Proteica , PrótonsRESUMO
A patient with nephrotic syndrome had been treated with mesalazine for ulcerative colitis, and also had a carcinoma of the colon and sclerosing cholangitis. This seemingly unrelated series of complications gives us the opportunity to review the problem of nephropathy in ulcerative colitis.
