RESUMO
The interaction of hemin with the first 18-amino acid repeat in tau protein has been investigated at both the N-terminal free-amine (R1τ) and N-acetylated (AcR1τ) forms for its potential relevance in traumatic brain injury and possibly other neurodegenerative diseases. The binding properties of hemin-R1τ and hemin-AcR1τ were compared with those of the hemin complex with amyloid-ß peptide fragment 1-16 (Aß16) and synthetic hemins. AcR1τ and R1τ bind with moderate affinity to both monomeric and dimeric hemin to form 1 : 1 complexes, but for the acetylated peptide, the affinity is one order of magnitude larger (K1 = 3.3 × 10(6) M(-1)). The binding constants were similar to that of Aß16 for hemin, but unlike the latter, neither of the two R1τ peptides forms a 2 : 1 complex with hemin. This is mostly due to electrostatic repulsion between R1τ chains, and in particular the C-terminal proline-15 kink, while structural features of the hemin-R1τ complexes do not seem to play a role. In fact, the same features are observed for the interaction between ferric heme and peptide R1τ*, where the P15 residue is replaced by an alanine. Imidazole neither binds to [hemin(R1τ)] nor [hemin(AcR1τ)], whereas small ligands such as CN and CO easily bind to the ferric and ferrous forms of the complexes, respectively. A detailed comparative study of the peroxidase activity of [hemin(R1τ)] and [hemin(AcR1τ)] shows that such activity is very low. Thus, the association between heme and unfolded neuronal peptides does not, per se, involve a significant gain of toxic pseudo-enzymatic activity. However, under conditions of heavy heme release occurring on traumatic brain injury or when this activity is prolonged for long time, it can contribute to neuronal oxidative stress. In addition, the presence of hemin increases the aggregation propensity of R1τ.
Assuntos
Heme/química , Peptídeos/química , Proteínas tau/química , Peróxido de Hidrogênio/química , Cinética , Oxirredução , Ligação ProteicaRESUMO
The minimal active domain (GEF domain) of the mouse Ras exchange factor CDC25Mm was purified to homogeneity from recombinant Escherichia coli culture. The 256 amino acids polypeptide shows high activity in vitro and forms a stable complex with H-ras p21 in absence of guanine nucleotides. Circular dichroism (CD) spectra in the far UV region indicate that this domain is highly structured with a high content of alpha-helix (42%). Near UV CD spectra evidenced good signal due to phenylalanine and tyrosine while a poor contribution was elicited by the three tryptophan residues contained in this domain. The tryptophan fluorescence signal was scarcely affected by denaturation of the protein or by formation of the binary complex with H-ras p21, suggesting that the Trp residues, which are well conserved in the GEF domain of several Ras-exchange factors, were exposed to the surface of the protein and they are not most probably directly involved in the interaction with Ras proteins.
Assuntos
Proteínas de Ciclo Celular/química , Fosfoproteínas Fosfatases/química , Estrutura Secundária de Proteína , Proteínas ras/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Dicroísmo Circular , Escherichia coli , Camundongos , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Alinhamento de Sequência , ras-GRF1RESUMO
The binding of hemin to the primary site of human serum albumin (HSA) has been reinvestigated using UV-Vis, CD and NMR techniques. The major fraction of bound hemin contains a five-coordinated high-spin iron(III) center, but a minor fraction of the metal appears to be in a six-coordinated, low-spin state, where a 'distal' residue, possibly a second histidine residue, completes the coordination sphere. The reduced, iron(II) form of the adduct contains six-coordinated low-spin heme. The distal residue hinders the access to the iron(III) center of hemin-HSA to small anionic ligands like azide and cyanide and destabilizes the binding of neutral diatomics like dioxygen and carbon monoxide to the iron(II) form. In spite of these limitations, the hemin-HSA complex promotes hydrogen peroxide activation processes that bear the characteristics of enzymatic reactions and may have biological relevance. The complex is in fact capable of catalyzing peroxidative reactions on phenolic compounds related to tyrosine and hydrogen peroxide dismutation. Kinetic and mechanistic studies confirm that the low efficiency with which peroxidative processes occur depends on the limited rate of the reaction between hydrogen peroxide and the iron(III) center, to form the active species, and by the competitive peroxide degradation reaction.
Assuntos
Hemina/química , Fenóis/química , Albumina Sérica/química , Catalase/química , Catálise , Dicroísmo Circular , Hemina/metabolismo , Homeostase , Humanos , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Químicos , Modelos Teóricos , Peroxidase/química , Albumina Sérica/metabolismo , EspectrofotometriaRESUMO
The chloroperoxidase catalyzed oxidation of methyl phenyl sulfide to (R)-methyl phenyl sulfoxide was investigated, both in batch and membrane reactors, using as oxidant H2O2, or O2 in the presence of either dihydroxyfumaric acid or ascorbic acid. The effects of pH and nature and concentration of the oxidants on the selectivity, stability, and productivity of the enzyme were evaluated. The highest selectivity was displayed by ascorbic acid/O2, even though the activity of chloroperoxidase with this system was lower than that obtained with the others. When the reaction was carried out in a membrane reactor, it was possible to reuse the enzyme for several conversion cycles. The results obtained with ascorbic acid/O2 and dihydroxyfumaric acid/O2 as oxidants do not seem to be compatible with either a mechanism involving hydroxyl radicals as the active species or with the hypothesis that oxidation occurs through the initial formation of H2O2. Copyright 1999 John Wiley & Sons, Inc.
RESUMO
Microperoxidase-8 (MP8) and microperoxidase-9 (MP9) have been covalently modified by attachment of proline-containing residues to the amino terminal peptide chain in order to obtain new peroxidase model systems. The catalytic activities of these derivatives in the oxidation of p-cresol by hydrogen peroxide have been compared to that of MP8. The presence of steric hindrance above the heme reduces the formation rate of the catalytically active species, while the reactivity is increased when the amino group of a proline residue is close to the iron. The modification of the catalyst affects the rate of degradation processes undergone by the heme group during catalysis. A bulky aromatic group on the distal side decreases the stability of the complex because it reduces the mobility of a phenoxy radical species formed during catalysis, while the presence of proline residues increases the number of turnovers of the heme catalysts before degradation. The complex Pro2-MP8 obtained by addition of two proline residues to MP8 exhibits the best catalytic performance in terms of activity and chemical stability.
Assuntos
Oligopeptídeos/química , Peroxidases/química , Peroxidases/metabolismo , Sequência de Aminoácidos , Animais , Grupo dos Citocromos c/química , Grupo dos Citocromos c/metabolismo , Heme/química , Cavalos , Cinética , Modelos Químicos , Conformação ProteicaRESUMO
Beta-endorphin, cholecystokinin and vasoactive intestinal peptide were measured in peripheral blood mononuclear cells of healthy controls, and schizophrenic patients at the first diagnosis before any treatment and after 2 or 15 d of treatment with haloperidol. Beta-endorphin concentrations were similar in controls and untreated patients, and increased with treatment. Cholecystokinin concentrations were higher in patients than in controls, and decreased during treatment. Vasoactive intestinal peptide was lower in patients and did not change with treatment. These observations are consistent with measurements of the same peptides in autopsy samples or cerebrospinal fluid. Peripheral blood mononuclear cells might be an useful tool for the study of some neuropeptides in brain.
Assuntos
Colecistocinina/sangue , Haloperidol/uso terapêutico , Monócitos/efeitos dos fármacos , Esquizofrenia/tratamento farmacológico , Psicologia do Esquizofrênico , Peptídeo Intestinal Vasoativo/sangue , beta-Endorfina/sangue , Adolescente , Adulto , Feminino , Humanos , Masculino , Monócitos/metabolismo , Escalas de Graduação Psiquiátrica , Esquizofrenia/sangue , Esquizofrenia/diagnósticoRESUMO
The lactoperoxidase (LPO)-catalyzed oxidation of p-phenols by hydrogen peroxide has been studied. The behavior of the enzyme differs from that of other peroxidases in this reaction. In particular LPO shows several catalytic intermediates during the catalytic cycle because of its capability to delocalize an oxidizing equivalent on a protein amino acid residue. In the phenol oxidation the enzyme Compound I species, containing an iron-oxo and a protein radical, uses the iron-oxo group at acidic pH and the protein radical in neutral or basic medium. Kinetic and spectroscopic studies indicate that the ionization state of an amino acid residue with pKa 5.8 +/- 0.2, probably the distal histidine, controls the enzyme intermediate forms at different pH. LPO undergoes inactivation during the oxidation of phenols. The inactivation is reversible and depends on the easy formation of Compound III even at low oxidant concentration. The inactivation is due to the substrate redox potential since the best substrate is that with lowest redox potential, while the worst substrate has the highest potential. This strongly indicates that Compound II, formed during catalytic turnover, has a low redox potential, making easier its oxidation by hydrogen peroxide to Compound III. The dependence of LPO activity on the phenols redox potential suggests that the protein radical where an oxidizing equivalent can be localized is a tyrosyl residue.
Assuntos
Lactoperoxidase/química , Fenóis/química , Catálise , Concentração de Íons de Hidrogênio , Oxirredução , Polarografia , Especificidade por SubstratoRESUMO
Competitive inhibition by phenolic compounds of the ascorbic acid oxidation reaction catalyzed by ascorbate oxidase was investigated at pH 7.0 and 23.0 degrees C. Inhibition of p-nitrophenol is pH dependent over the range 5.0-8.0, with inhibitor binding favored at higher pH. Bulky substituents on the phenol nucleus reduce or prevent the inhibitory effect. The presence of phenol affects the binding characteristics of azide to the trinuclear cluster of the enzyme. In particular, binding of azide to type 2 copper is prevented, and the affinity of azide to type 3 copper is reduced. In addition, reduction of type 1 copper is observed upon prolonged incubation of ascorbate oxidase with excess phenol and azide, but not with phenol alone. It is proposed that binding of phenolic inhibitors occurs at or near the site where the substrate (ascorbate) binds. NMR relaxation measurements of the protons of phenols in the presence of ascorbate oxidase show paramagnetic effects due to the proximity of the bound inhibitor to a copper center, likely type 1 copper. Copper-proton distance estimates between this paramagnetic center and p-cresol or p-nitrophenol bound to ascorbate oxidase are between 4.4 and 5.9 A.
Assuntos
Ascorbato Oxidase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fenóis/farmacologia , Ligação Competitiva , Clorofenóis/farmacologia , Cresóis/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Concentração de Íons de Hidrogênio , Nitrofenóis/farmacologia , Fenol , Espectrofotometria UltravioletaRESUMO
The present study was carried out to investigate the routine use of second-generation antipsychotic drugs in the Italian psychiatric care system. Seven outpatient psychiatric services enrolled a consecutive case series of patients who were being treated, or had started treatment, with clozapine, olanzapine, risperidone, or quetiapine. Information on sociodemographic and clinical variables, current psychotropic drug use, side-effects and past use of typical drugs was collected. In addition, patient symptoms and functional status were evaluated by the Health of the Nation Outcome Scale. Patients receiving off-label prescribing of second-generation antipsychotics were identified. A total of 209 patients were collected. In comparison with patients receiving other second-generation antipsychotics, living in residential facilities, unemployment, long psychiatric histories, and problems with activities of daily living and living conditions were more common in clozapine-treated patients. Nearly 80 % of patients receiving clozapine had schizophrenia compared to less than 50 % of those receiving other second-generation antipsychotics. Overall, 109 patients (52 %) received off-label prescriptions of second-generation antipsychotic drugs. This survey indicates that clozapine was mostly reserved for severe cases and poor responders; the high rate of off-label prescriptions highlights the gap existing between recommendations derived from randomised clinical trials and the current use of drugs.
Assuntos
Antidepressivos de Segunda Geração/uso terapêutico , Serviços Comunitários de Saúde Mental/tendências , Pirenzepina/análogos & derivados , Esquizofrenia/tratamento farmacológico , Adolescente , Adulto , Idoso , Benzodiazepinas , Escalas de Graduação Psiquiátrica Breve , Clozapina/uso terapêutico , Dibenzotiazepinas/uso terapêutico , Quimioterapia Combinada , Uso de Medicamentos/tendências , Feminino , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Olanzapina , Pacientes Ambulatoriais , Farmacoepidemiologia , Pirenzepina/uso terapêutico , Escalas de Graduação Psiquiátrica , Fumarato de Quetiapina , Risperidona/uso terapêutico , Esquizofrenia/diagnóstico , Resultado do TratamentoRESUMO
The inhibition of the catechol oxidase activity exhibited by three dinuclear copper(II) complexes, derived from different diaminotetrabenzimidazole ligands, by kojic acid [5-hydroxy-2-(hydroxymethyl)-gamma-pyrone] has been studied. The catalytic mechanism of the catecholase reaction proceeds in two steps and for both of these inhibition by kojic acid is of competitive type. The inhibitor binds strongly to the dicopper(II) complex in the first step and to the dicopper-dioxygen adduct in the second step, preventing in both cases the binding of the catechol substrate. Binding studies of kojic acid to the dinuclear copper(II) complexes and a series of mononuclear analogs, carried out spectrophotometrically and by NMR, enable us to propose that the inhibitor acts as a bridging ligand between the metal centers in the dicopper(II) catalysts.
Assuntos
Catecol Oxidase/antagonistas & inibidores , Cobre/química , Inibidores Enzimáticos/farmacologia , Micotoxinas/antagonistas & inibidores , Pironas/antagonistas & inibidores , Algoritmos , Benzimidazóis/química , Benzimidazóis/farmacologia , Catálise , Indicadores e Reagentes , Cinética , Ligantes , Espectroscopia de Ressonância Magnética , Micotoxinas/química , Pironas/química , Difração de Raios XRESUMO
The complex [Cu2(L-66)]2+ (L-66 = a,a'-bis¿bis[2-(1'-methyl-2'-benzimidazolyl)ethyl]amino¿-m-xylene) undergoes fully reversible oxygenation at low temperature in acetone. The optical [lambda(max) = 362 (epsilon 15000), 455 (epsilon 2000), and 550 nm (epsilon 900M(-1)cm(-1))] and resonance Raman features (760 cm(-1), shifted to 719cm(-1)(-1) with 18O2) of the dioxygen adduct [Cu2(L-66)(O2)]2+ indicate that it is a mu-eta2:eta2-peroxodicopper(II) complex. The kinetics of dioxygen binding, studied at - 78 degrees C, gave the rate constant k1 = 1.1M(-1) 5(-1) for adduct formation, and k(-1) =7.8 x 10(-5)s(-1), for dioxygen release from the Cu2O2 complex. From these values, the O2 binding constant K= 1.4 x 10(4)M(-1) at -78 degrees C could be determined. The [Cu2(L-66)(O2)]2+ complex performs the regiospecific ortho-hydroxylation of 4-carbomethoxyphenolate to the corresponding catecholate and the oxidation of 3,5-di-tert-butylcatechol to the quinone at -60 degrees C. Therefore, [Cu2(L-66)]2+ is the first synthetic complex to form a stable dioxygen adduct and exhibit true tyrosinase-like activity on exogenous phenolic compounds.
Assuntos
Modelos Químicos , Monofenol Mono-Oxigenase/metabolismo , Oxigênio/metabolismo , Fenóis/metabolismo , Oxirredução , Espectrofotometria AtômicaRESUMO
Tetraheme cytochrome c3 (13 kDa) and flavodoxin (16 kDa), are small electron transfer proteins that have been used to mimic, in vitro, part of the electron-transfer chain that operates between substract electron donors and respiratory electron acceptors partners in Desulfovibrio species (Palma, N., Moura, I., LeGall, J., Van Beeumen, J., Wampler, J., Moura, J. J. G. (1994) Biochemistry 33, 6394-6407). The electron transfer between these two proteins is believed to occur through the formation of a specific complex where electrostatic interaction is the main driving force (Stewart, D., LeGall, J., Moura, I., Moura, J.J.G., Peck, H.D., Xavier, A.V., Weiner, P.K. and Wampler, J.E. (1988) Biochemistry 27, 2444-2450, Stewart, D., LeGall, J., Moura, I., Moura, J.J.G., Peck, H.D., Xavier, A.V., Weiner, P., Wampler, J. (1989) Eur. J. Biochem. 185, 695-700). In order to obtain structural information of the pre-complex, a covalent complex between the two proteins was prepared. A water-soluble carbodiimide [EDC (1-ethyl-3(3 dimethylaminopropyl) carbodiimide hydrochloride] was used for the cross linking reaction. The reaction was optimized varying a wide number of experimental parameters such as ionic strength, protein and cross linker concentration, and utilization of different cross linkers and reaction time between the crosslinker and proteins.
Assuntos
Reagentes de Ligações Cruzadas , Grupo dos Citocromos c/metabolismo , Desulfovibrio/metabolismo , Flavodoxina/metabolismo , CME-Carbodi-Imida/análogos & derivados , Cromatografia Líquida de Alta Pressão , Grupo dos Citocromos c/química , Relação Dose-Resposta a Droga , Transporte de Elétrons , Etildimetilaminopropil Carbodi-Imida , Flavodoxina/química , Concentração de Íons de Hidrogênio , Peso Molecular , Concentração Osmolar , Ligação Proteica/efeitos dos fármacos , Cloreto de Sódio/farmacologia , Eletricidade Estática , Succinimidas , Fatores de TempoRESUMO
The cellular prion protein (PrPC) is a sialoglycoprotein of M(r) 33-35K that is expressed predominantly in neurons. In transmissible and genetic neurodegenerative disorders such as scrapie of sheep, spongiform encephalopathy of cattle and Creutzfeldt-Jakob or Gerstmann-Sträussler-Scheinker diseases of humans, PrPC is converted into an altered form (termed PrPSc) which is distinguishable from its normal homologue by its relative resistance to protease digestion. PrPSc accumulates in the central nervous system of affected individuals, and its protease-resistant core aggregates extracellularly into amyloid fibrils. The process is accompanied by nerve cell loss, whose pathogenesis and molecular basis are not understood. We report here that neuronal death results from chronic exposure of primary rat hippocampal cultures to micromolar concentrations of a peptide corresponding to residues 106-126 of the amino-acid sequence deduced from human PrP complementary DNA. DNA fragmentation of degenerating neurons indicates that cell death occurred by apoptosis. The PrP peptide 106-126 has a high intrinsic ability to polymerize into amyloid-like fibrils in vitro. These findings indicate that cerebral accumulation of PrPSc and its degradation products may play a role in the nerve cell degeneration that occurs in prion-related encephalopathies.
Assuntos
Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/toxicidade , Príons/toxicidade , Sequência de Aminoácidos , Animais , Apoptose , Células Cultivadas , Relação Dose-Resposta a Droga , Hipocampo/citologia , Hipocampo/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , RatosRESUMO
OBJECTIVE: Following the introduction of guidelines of rational drug use, the pharmacoepidemiology of psychotropic drugs was investigated in a sample of long-stay patients living in a Italian psychiatric hospital. DESIGN: A prospective, longitudinal two-year follow-up study was carried out. Information about sociodemographic and clinical characteristics of the inpatient population, and about medications prescribed, was collected at baseline and after one and two years of follow-up. SETTING: Three wards of the psychiatric hospital of Milan. MAIN OUTCOME MEASURES: Number of patients taking psychotropic drugs, number of patients taking more than one neuroleptic or benzodiazepine, mean neuroleptic dose, psychopathological status according to the Brief Psychiatric Rating Scale (BPRS). RESULTS: 70 patients were recruited and followed for two years. At follow-up a reduction in the number of patients taking neuroleptic drugs was recorded, together with a 50% decrease in the number of patients taking more than one neuroleptic. A reduction in the use of depot formulations was in addition shown. Patients taking benzodiazepines decreased of 50%. According to the BPRS, no psychopatological changes were observed during the study. CONCLUSIONS: These data suggest that education in psychopharmacology may guide towards a more rational use of drugs; longitudinal clinical audits should be implemented to monitor everyday practice.
Assuntos
Hospitais Psiquiátricos/estatística & dados numéricos , Transtornos Mentais/tratamento farmacológico , Psicotrópicos/uso terapêutico , Adulto , Idoso , Escalas de Graduação Psiquiátrica Breve , Área Programática de Saúde , Uso de Medicamentos , Feminino , Seguimentos , Humanos , Itália , Masculino , Transtornos Mentais/diagnóstico , Transtornos Mentais/reabilitação , Pessoa de Meia-IdadeRESUMO
Dinuclear copper(II) complexes with N-substituted sulfonamide ligands as superoxide dismutase (SOD) mimics have been investigated. The new N-(thiazol-2-yl)toluenesulfonamide (Htz-tol) and N-(thiazol-2-yl)naphthalenesulfonamide (Htz-naf) ligands have been prepared and structurally characterized. The complexes derived from these ligands, [Cu(2)(tz-tol)(4)] (1) and [Cu(2)(tz-naf)(4)] (2), have been synthesized, and their crystal structure, magnetic properties, and EPR spectra were studied in detail. In both compounds the metal centers are bridged by four nonlinear triatomic NCN groups. The coordination geometry of the coppers in the dinuclear entity of 1 and 2 is distorted square planar with two N-thiazole and two N-sulfonamido atoms. Magnetic susceptibility data show a strong antiferromagnetic coupling, with -2J = 121.3 cm(-1) for compound 1 and -2J = 104.3 cm(-1) for compound 2. The EPR spectra of the polycrystalline samples of compounds 1 and 2 have been measured at the X- and Q-band frequencies at different temperatures. Above 20 K the spectra are characteristic of S = 1 species with zero-field splitting parameter D = 0.230 cm(-1) for compound 1 and 0.229 cm(-1) for compound 2. The EPR parameters are discussed in terms of the known binuclear structures. The complexes exhibit high SOD activity, as shown by the low IC(50) values obtained with the xanthine/xanthine oxidase/NBT assay: 0.13 microM for compound 1; 0.17 microM for compound 2.
Assuntos
Cobre/química , Compostos Organometálicos/síntese química , Superóxido Dismutase/química , Algoritmos , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Ligantes , Magnetismo , Modelos Moleculares , Conformação Molecular , Mimetismo Molecular , Estrutura Molecular , Compostos Organometálicos/química , Superóxido Dismutase/metabolismoRESUMO
The properties of type I collagen CNBr peptides in solution were studied to investigate the molecular species formed, their conformation, and factors influencing equilibria between peptide species. Peptides formed homologous trimers, even though the native parent protein is heterotrimeric, [alpha 1(I)]2 alpha 2-(I). Their triple-helical content was found to be high (> 75% for most peptides). Full helical content was not reached mainly because of the presence of monomer species; chain misalignment, if present, and trimer unraveling at terminal ends appeared to play a minor role in reducing helicity. Circular dichroism spectra and resistance to trypsin digestion at 4 and 20 degrees C demonstrated that the conformation of trimers was very similar to the collagen triple-helical conformation. Rotary shadowing of peptide alpha 1(I) CB7 supported this finding. Analytical gel filtration in nondenaturing conditions showed that the trimers of some peptides have the ability to autoaggregate. In the case of peptides alpha 1(I) CB8 and alpha 2(I) CB4, most of the intermolecular interactions between trimeric molecules were disrupted by 0.5 M NaCl, demonstrating that their ionic character is important. Changes in ionic strength also altered the hydrodynamic size of single- and triple-stranded molecules. The different molecular species are in equilibrium. The kinetics of the conversion of trimer to monomer species was determined in a time course experiment using trypsin digestion and found to be a relatively slow process (trimer half-life is a few days at 4 degrees C, about one order of magnitude lower at 20 degrees C) with an activation energy of roughly 4-9 kcal/mol. The circular dichroism profile at increasing temperatures showed that the melting temperature for triple-helical peptides is about 6-10 degrees C lower than that of the parent native type I collagen. The folding of peptides is a spontaneous process (exothermic but with unfavourable entropy change), and the triple-helical conformation originates solely as the result of the collagen sequence because it forms from heat-denatured samples.
Assuntos
Colágeno/química , Fragmentos de Peptídeos/química , Animais , Bovinos , Cromatografia em Gel , Cromatografia por Troca Iônica , Dicroísmo Circular , Colágeno/metabolismo , Brometo de Cianogênio , Hidrólise , Cinética , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Tripsina/metabolismoRESUMO
The synthetic complexes protohemin-6(7)-L-arginyl-L-alanine (HM-RA) and protohemin-6(7)-L-histidine methyl ester (HM-H) were prepared by condensation of suitably protected Arg-Ala or His residues with protohemin IX. HM-RA and HM-H were used for reconstitution of apomyoglobin from horse heart, yielding the Mb-RA and Mb-H derivatives, respectively, of the protein. The spectral, binding and catalytic properties of Mb-RA and Mb-H are significantly different from those of Mb. As shown by MM and MD calculations, these differences are determined by some local structural changes around the heme which are generated by increased mobility of a key peptide segment (Phe43-Lys47), containing the residue (Lys45) that in native Mb interacts with one of the porphyrin carboxylate groups. In the reconstituted Mbs this carboxylate group is bound to the Arg-Ala or His residue and is no longer available for electrostatic interaction with Lys45. The mobility of the peptide segment near the active site allows the distal histidine to come to a closer contact with the heme, and in fact Mb-RA and Mb-H exist as an equilibrium between a high-spin form and a major low-spin, six-coordinated form containing a bis-imidazole ligated heme. The two forms are clearly distinguishable in the NMR spectra, that also show that each of them consists of a mixture of the two most stable isomers resulting from cofactor reconstitution, as also anticipated by MM and MD calculations. Exogenous ligands such as cyanide, azide, or hydrogen peroxide can displace the bound distal histidine, but their affinity is reduced. On the other hand, mobilization of the peptide chain around the heme in the reconstituted Mbs increases the accessibility of large donor molecules at the heme periphery, with respect to native Mb, where a rigid backbone limits access to the distal pocket. The increased active site accessibility of Mb-RA and Mb-H facilitates the binding and electron transfer of phenolic substrates in peroxidase-type oxidations catalyzed by the reconstituted proteins in the presence of hydrogen peroxide.
Assuntos
Hemina/química , Mioglobina/química , Alanina/química , Alanina/metabolismo , Animais , Apoproteínas/metabolismo , Arginina/química , Arginina/metabolismo , Sítios de Ligação , Dipeptídeos/química , Dipeptídeos/metabolismo , Hemina/síntese química , Hemina/metabolismo , Histidina/química , Histidina/metabolismo , Cavalos , Cinética , Ligantes , Mioglobina/metabolismo , Conformação ProteicaRESUMO
The reactivity of nitrite towards the copper(II) and copper(I) centers of a series of complexes with tridentate nitrogen donor ligands has been investigated. The ligands are bis[(1-methylbenzimidazol-2-yl)methyl]amine (1-bb), bis[2-(1-methylbenzimidazol-2-yl)ethyl]amine (2-bb), and bis[2-(3,5-dimethyl-1-pyrazolyl)ethyl]amine (ddah) and carry two terminal benzimidazole (1-bb, 2-bb) or pyrazole (ddah) rings and a central amine donor residue. While 2-bb and ddah form two adjacent six-membered chelate rings on metal coordination, 1-bb forms two smaller rings of five members. The binding affinity of nitrite and azide to the Cu(II) complexes (ClO4- as counterion) has been determined in solution. The association constants for the two ligands are similar, but nitrite is a slightly stronger ligand than azide when it binds as a bidentate donor. The X-ray crystal structure of the nitrite complex [Cu(ddah)(NO2)]ClO4 (final R=0.056) has been determined: triclinic P1space group, a=8.200(2) A, b=9.582(3) A, c=15.541(4) A. It may be described as a perchlorate salt of a "supramolecular" species resulting from the assembly of two complex cations and one sodium perchlorate unit. The copper stereochemistry in the complex is intermediate between SPY and TBP, and nitrite binds to Cu(II) asymmetrically, with Cu-O distances of 2.037(2) and 2.390(3) A and a nearly planar CuO2N cycle. On standing, solutions of [Cu(ddah)(NO2)]ClO4 in methanol produce the dinuclear complex [Cu(ddah)(OMe)]2(ClO4)2, containing dibridging methoxy groups. In fact the crystal structure analysis (final R=0.083) showed that the crystals are built up by dinuclear cations, arranged on a crystallographic symmetry center, and perchlorate anions. Electrochemical analysis shows that binding of nitrite to the Cu(II) complexes of 2-bb and ddah shifts the reduction potential of the Cu(II)/Cu(I) couple towards negative values by about 0.3 V. The thermodynamic parameters of the Cu(II)/Cu(I) electron transfer have also been analyzed. The mechanism of reductive activation of nitrite to nitric oxide by the Cu(I) complexes of 1-bb, 2-bb, and ddah has been studied. The reaction requires two protons per molecule of nitrite and Cu(I). Kinetic experiments show that the reaction is first order in [Cu(I)] and [H+] and exhibits saturation behavior with respect to nitrite concentration. The kinetic data show that [Cu(2-bb)]+ is more efficient than [Cu(1-bb)]+ and [Cu(ddah)]+ in reducing nitrite.