共查询到20条相似文献,搜索用时 15 毫秒
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Friedecký D Bednár P Procházka M Adam T 《Nucleosides, nucleotides & nucleic acids》2006,25(9-11):1233-1236
A pilot study using capillary electrophoresis with mass spectrometry for the analysis of nucleotides in human erythrocytes is presented. Erythrocytes were incubated with 5-amino-4-imidazolecarboxamide riboside in order to mimic situation in defect of purine metabolism--AICA-ribosiduria. Characteristic AICA-ribotides together with normal nucleotides were separated by capillary electrophoresis in acetate buffer (20 mmol/L, pH 4.4) and identified on line by mass spectrometry. 相似文献
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Molecular dynamics study of Desulfovibrio africanus cytochrome c3 in oxidized and reduced forms
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A 5-ns molecular dynamics study of a tetraheme cytochrome in fully oxidized and reduced forms was performed using the CHARMM molecular modeling program, with explicit water molecules, Langevin dynamics thermalization, Particle Mesh Ewald long-range electrostatics, and quantum mechanical determination of heme partial charges. The simulations used, as starting points, crystallographic structures of the oxidized and reduced forms of the acidic cytochrome c(3) from Desulfovibrio africanus obtained at pH 5.6. In this paper we also report structures for the two forms obtained at pH 8. In contrast to previous cytochrome c(3) dynamics simulations, our model is stable. The simulation structures agree reasonably well with the crystallographic ones, but our models show higher flexibility and the water molecules are more labile. We have compared in detail the differences between the simulated and experimental structures of the two redox states and observe that the hydration structure is highly dependent on the redox state. We have also analyzed the interaction energy terms between the hemes, the protein residues, and water. The direct electrostatic interaction between hemes is weak and nearly insensitive to the redox state, but the remaining terms are large and contribute in a complex way to the overall potential energy differences that we see between the redox states. 相似文献
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Hydrogen-deuterium exchange in 2H20 solutions of the two redox states of horse heart cytochrome c was investigated at 20 degrees C, pH 7, by mass spectrometry and infrared spectroscopy. Mass spectrometry indicates that ferricytochrome has 20 hydrogens unexchanged after 24 h, 28 hydrogens exchanging between 10 min and 24 h, and 156 hydrogens exchanging within 10 min; comparative values for ferrocytochrome are 45, 19 and 140. The displacement of the exchange curves obtained by infrared corresponds to 8 to 9 peptide hydrogens. These combined methods show many non-peptide hydrogens exchanging rapidly (87 and 79 for ferricytochrome c and ferrocytochrome c respectively), whereas others, probably buried inside the molecule and involved in hydrogen bonds, are not exchanged, even after 24 h (14 and 30 hydrogens respectively, which is relatively large for a small protein). Infrared results are given in terms of changes of standard free energy for the transconformational reaction which exposes the peptide hydrogens to solvent: in ferricytochrome c and ferrycoytochrome c, 30% and 40% respectively of the peptide hydrogens are protected by conformational transitions stabilized by more than 5 kcal/mol (21 kJ/mol), which implies a large increase in rigidity for the reduced form. 相似文献
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Chromatography of oxidized and reduced cytochrome c on carboxymethylcellulose 总被引:1,自引:1,他引:0
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1. Cytochrome c was isolated from horse heart by a chromatographic method. 2. Oxidized and reduced cytochrome c were chromatographed on CM-cellulose that was in equilibrium with several buffer systems of constant composition at pH values of 8.4, 6.75 and 4.9. 3. Separation was better at the higher pH values; the oxidized form was retarded more than twice as much as the reduced form, though they differed by only a single charge. 4. Self-competition between cytochrome molecules is suggested to account for the peak distortion observed at high loads (above 20mum protein concentration). 相似文献
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S Rosén 《Biochimica et biophysica acta》1978,503(2):389-397
1. Kinetic studies have been performed with beef-heart cytochrome c oxidase, with the enzyme either in its oxidized, resting state or pretreated anaerobically with different amounts of reduced cytochrome c. The techniques used for the study have been stopped-flow spectrophotometry and electron paramagnetic resonance (EPR) spectroscopy. 2. The results show that the one-electron equivalent-reduced enzyme rapidly oxidizes one further equivalent of aerobically or anaerobically added ferrocytochrome c, with a rate constant of 5 . 10(6) M-1 . s-1. 3. When an excess of ferrocytochrome c in the presence of oxygen is added to the one-electron-reduced enzyme, the same turnover rate is obtained as in experiments with the resting enzyme. 4. The one-electron equivalent-enzyme reacts with CO with a rate constant of 4 . 10(4) M-1 . s-1 to yield approx. 35% of the CO compound as compared with the reaction between the fully reduced enzyme and CO. 5. It is shown that on reduction the enzyme is converted into an active form, but it is concluded that the enzyme does not have to be fully reduced before it is catalytically active. 相似文献
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Weixia Liu Jon N. Rumbley S. Walter Englander A. Joshua Wand 《Protein science : a publication of the Protein Society》2009,18(3):670-674
The sub‐nanosecond structural dynamics of reduced and oxidized cytochrome c were characterized. Dynamic properties of the protein backbone measured by amide 15N relaxation and side chains measured by the deuterium relaxation of methyl groups change little upon change in the redox state. These results imply that the solvent reorganization energy associated with electron transfer is small, consistent with previous theoretical analyses. The relative rigidity of both redox states also implies that dynamic relief of destructive electron transfer pathway interference is not operational in free cytochrome c. 相似文献
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M Assfalg L Banci I Bertini M Bruschi M T Giudici-Orticoni P Turano 《European journal of biochemistry》1999,266(2):634-643
The solution structure via 1H NMR of the fully reduced form of cytochrome c7 has been obtained. The protein sample was kept reduced by addition of catalytic amounts of Desulfovibrio gigas iron hydrogenase in H2 atmosphere after it had been checked that the presence of the hydrogenase did not affect the NMR spectrum. A final family of 35 conformers with rmsd values with respect to the mean structure of 8.7 +/- 1.5 nm and 12.4 +/- 1.3 nm for the backbone and heavy atoms, respectively, was obtained. A highly disordered loop involving residues 54-61 is present. If this loop is ignored, the rmsd values are 6.2 +/- 1.1 nm and 10.2 +/- 1.0 nm for the backbone and heavy atoms, respectively, which represent a reasonable resolution. The structure was analyzed and compared with the already available structure of the fully oxidized protein. Within the indetermination of the two solution structures, the result for the two redox forms is quite similar, confirming the special structural features of the three-heme cluster. A useful comparison can be made with the available crystal structures of cytochromes c3, which appear to be highly homologous except for the presence of a further heme. Finally, an analysis of the factors affecting the reduction potentials of the heme irons was performed, revealing the importance of net charges in differentiating the reduction potential when the other parameters are kept constant. 相似文献
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C. Muscari M. Pappagallo D. Ferrari E. Giordano C. Capanni C. M. Caldarera C. Guarnieri 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1998,707(1-2)
We have developed a rapid and precise method for glutathione quantitation by capillary electrophoresis, that allows a low amount of both redox forms to be measured. Small fragments of rat heart or liver tissues (20 mg wet weight) and the corresponding mitochondria (1 mg protein) were homogenized in 1% perchloric acid and the acid-soluble phase ultrafiltered by centrifugation with a microconcentrator (Mr cut-off 3000 Da). The analysis was performed at a constant temperature (28°C) using a Beckman P/ACE System 2100, equipped with a UV absorbance detector set to 200 nm. The limit of quantitation in heart tissue was 1.8 μM for GSH and 1.2 μM for GSSG. Myocardial concentrations of GSH and GSSG were 8.1±2.6 and 0.45±0.15 (nmol/mg protein±S.D.), respectively. The ratio of GSH to GSSG was 17.8±1.3 for heart tissue, whereas it was much higher (>100) in the mitochondria. An oxidative stress decreased the myocardial tissue GSH/GSSG ratio, indicating that the CE analysis of both glutathione forms is also a useful method to study biological redox modification. 相似文献
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Peptide and protein analysis by electrospray ionization-mass spectrometry and capillary electrophoresis-mass spectrometry 总被引:3,自引:0,他引:3
The extension of mass spectrometry to high molecular weight biopolymers based upon electrospray ionization and the on-line combination with capillary electrophoresis is described. Electrospray ionization produces gas-phase intact multiply charged molecular ions of biomolecules from highly charged liquid droplets by a high electric field. For high molecular weight substances electrospray ionization results in a characteristic bell-shaped distribution of multiply charged ions, with each adjacent major peak in the spectrum differing by one charge. Multiply charged molecular ions of proteins with molecular weights greater than 130,000 have been observed with a quadrupole mass spectrometer of limited mass-to-charge range (m/z 1700). Molecular weights can be readily determined for large proteins with accuracies in the range of +/- 0.01 to 0.05%; at least an order of magnitude further improvement appears feasible with improved techniques and instrumentation. The electrospray ionization method is sensitive, presently requiring samples in the 100 fmol to 10 pmol range for proteins. Initial results combining rapid separations by capillary zone electrophoresis with on-line mass spectrometric detection via the electrospray ionization source are demonstrated for myoglobin and other proteins and polypeptides. The potential for extension of these methods to molecular weights on the order of 10(6) is discussed. 相似文献
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The surface properties of monomolecular films of oxidized and reduced cytochromes f and c were measured at an air-water interface. Area/molecular (A) and surface potential (deltaV) for oxidized and reduced forms of the cytochromes were measured as a function of pH. Oxidized cyt f has a maximum for both A and deltaV at pH 7.5. At a surface pressure of 6 dyn/cm the maximum A equals 2600 plus or minus 50 A2 and the maximum deltaV equals 200 plus or minus 10 mV. Reduced cyt f as a function of pH has a minimum value for both A (2200 A2) and deltaV (95 mV). Oxidized cyt c as a function of pH has minima for A (140 A2) and deltaV (188 mV) at pH 7.0 and 7.3, respectively. On the other hand, reduced cyt has maximum values for A (220 A2) and deltaV (260 mV) at pH 7.0 and 7.3, respectively. 相似文献
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Potentiometric titration curves of oxidized and reduced horse heart cytochrome c in 0.15M KCl at 20°C have been obtained by timed titration (0.125–0.500 μmol/sec) from the isoionic points (pH 10.2–10.4) to pH 3 and back to the isoionic point. Computer-assisted (PROPHET) data acquisition and blank corrections give curves with good precision with a maximum standard deviation of 0.3 groups for an average error of 1%. The potentiometric titration curve of reduced cytochrome c is reversible within the precision of the method and for the pH range studied. The potentiometric curves for oxidized cytochrome c titrated upscale (pH 3–10) and downscale (pH 10–3) are not reversible. However, they show the same ionization behavior after the initial downscale titration. This is probably the result of a conformational change. Comparison of the data herein reported with the titration curves of oxidized cytochrome c already published by others indicates good agreement on the basis of a normalization of the concentration of protein or on the basis of 25 titrable groups between the acid end point and the isoionic pH. Titration of the 2 μmol imidazole in the upscale or downscale direction gives the correct analytical concentration and pK′ after correction for the solvent titration. Titration of reduced cytochrome c in the presence and absence of an additional equivalent of imidazole gave a difference titration curve, which indicates that a group on the protein shifts from pK′ 5.8 to pK′ 5.3 in the presence of imidazole. The pK′ of imidazole, in the presence of the protein, remains at a nearly normal value of 7.34. 相似文献
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Glycosylation is a complex posttranslational modification that can result in extensive heterogeneity for recombinant glycoproteins
produced by eukaryotic systems. The carbohydrate moiety of a recombinant glycoprotein may affect the immunogenicity, half-life,
bioactivity, and stability of a potential therapeutic product. Regulatory authorities such as the US Food and Drug Administration
demand increasingly sophisticated carbohydrate analysis to ensure product characterization, batch-to-batch consistency, and
stability.
The advent of new technologies for analysis of biopolymers by capillary electrophoresis and mass spectrometry has revolutionized
strategies for recombinant protein characterization. In particular, recent advances in matrix-assisted laser desorption/ionization
and electrospray ionization mass spectrometry now permit relatively rapid and detaned assessment of glycoprotein and oligosaccharide
structure. In this article, we describe some applications of capillary electrophoresis and mass spectrometry to monitor the
glycosylation associated with a model recombinant glycoprotein, human interferon-γ. 相似文献
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Analysis of lipophilic peptides and therapeutic drugs: on-line-nonaqueous capillary electrophoresis-mass spectrometry 总被引:4,自引:0,他引:4
Yang Q Benson LM Johnson KL Naylor S 《Journal of biochemical and biophysical methods》1999,38(2):103-121
This minireview addresses the usefulness of nonaqueous capillary electrophoresis-mass spectrometry (NACE-MS), mainly in the analysis of lipophilic peptides such as gramicidin S and bacitracin, and therapeutic drugs such as pyrazoloacridine, the H2-antagonist mifentidine, tamoxifen, and their metabolites. The beneficial effects of NACE-MS in typical bioanalytical applications are analyzed case by case. A suitable and widely applicable NACE-MS analysis is identified, which is an electrolyte buffer containing ammonium acetate (5-50 mM) and/or acetic acid (up to 100 mM) with varying composition of organic solvents. Either acetonitrile or methanol or a mixture of the two are mostly utilized in the nonaqueous media. Primary considerations in developing NACE-MS are also discussed. 相似文献
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Analysis of the ionization constants and heats of ionization of reduced and oxidized horse heart cytochrome c 总被引:1,自引:0,他引:1
Simultaneous curve fitting for the ionization parameters of oxidized and reduced horse heart cytochrome c in 0.15M KCl and 20°C yields values for the ionization constants (as pK′) and the heats of ionization (ΔHi) which can reconstruct either the potentiometric or thermal titration curves. Reduced cytochrome c requires 8 sets of groups, whereas oxidized cytochrome c requires 10 sets of groups. The additional groups in the oxidized preparation appear to involve the ferriheme (pK′, 9.25; ΔHi, 13.7 kcal/mol) and a tyrosine (pK′ ? 10.24) that is not present in the reduced form. The potentiometric and thermal difference curves (reduced – oxidized) involve the appearance of 17 kcal/mol centered at pH 9.7 and 5.8 kcal/mol centered at pH 4.9. The carboxyl groups in both species appear to be normal for the hydrogen-bonded form. Only one histidine has normal ionization properties (pK′, 6.7; ΔHi, 7.5 kcal/mol), as do 17 of the lysine residues (pK′, 10.8; ΔHi, 11.5 kcal/mol). 相似文献
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Bovine heart mitochondrial cytochrome c oxidase (cytochrome aa3) (EC 1.9.3.1) has been demonstrated to occur in several forms when the redox centers in the protein are thought to be fully oxidized. We report here the results of extensive EPR studies at 3, 8.9, 9.2, 9.4, 15 and 34 GHz on the resting state, the alternative resting state (with g = 12 at 9 GHz) and pulsed state (with g = 5 signal at 9 GHz). Theoretical consideration is given to all binary spin-coupling possibilities under the constraint that the iron atoms are either ferric or ferrous and the copper atoms are either cupric or cuprous. We conclude that the g = 12 signal can arise from any spin system with S greater than 1 and D = 0.15 cm-1. The g = 5 signals originate from an excited, integer-spin system with D = 0.035 cm-1, which is approximately 7 cm-1 above the ground state (not observed in EPR). It is pointed out that in interpretations of data and elaboration of suitable models in this field, the implications of spin-coupling should be considered in a comprehensive and not in a selective way. At 3 GHz, EPR spectra of CuA in the resting, pulsed and anaerobically oxidized states show that this center is identical in its EPR for all three states. 相似文献
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Differences in the solution structures of oxidized and reduced cytochrome c measured by small-angle X-ray scattering 总被引:1,自引:0,他引:1
While X-ray crystallographic data on cytochrome c show the reduced and oxidized forms to have very similar structures, there is a considerable body of data, mostly from solution studies, that indicates the reduced form is more stable and that the interior of the protein is less accessible to solvent in this state. These observations have led to the hypothesis that while the time-averaged structure is preserved between the two forms, the dynamics of the two forms are different. The oxidized form has been proposed to undergo more large-amplitude, low-frequency motions than the reduced form. The crystal structure data were derived from crystals grown in high salt concentrations, but the solution studies were done at relatively low ionic strength. Small-angle X-ray scattering has been used to examine the effects of the ionic strength and oxidation state on the solution structure of cytochrome c. We find that the radius of gyration and the maximum linear dimension of oxidized cytochrome c are significantly larger than those for reduced cytochrome c, in 5 mM phosphate buffer at pH 7.3, and further that this difference is suppressed by addition of 200 mM sodium chloride. We conclude that there is a real structural difference between the two forms at low ionic strength in solution and that this difference is likely to contribute to the observed differences in accessibility and compressibility. 相似文献