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PubMed2000

Optical characterization of liposomes by right angle light scattering and turbidity measurement.

Liposomes have frequently been used as models of biomembranes or vehicles for drug delivery. However, the systematic characterization of lipid vesicles by right angle light scattering and turbidity has not been carried out despite the usefulness of such studies for size estimation. In this study, liposomes of various sizes were prepared by sonication and extrusion. The mean cumulant radii of the vesicles were determined by dynamic light scattering. The lamellarities were estimated based on fluorescence quenching of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L-alpha-phosph ati dylethanolamine by sodium dithionite. Right angle light scattering intensity and optical density at 436 nm per unit lipid concentration were measured as a function of vesicle radius. With a vesicle radius < or =100 nm, the optical parameters could be well explained by the Rayleigh-Gans-Debye theory in which the liposomes were modeled as homogeneous spheres with mean refractive indices determined by the volume fractions of lipids in vesicles.

Dithionite↗
PubMed1997

Fluorescence studies on the interaction of a synthetic signal peptide and its analog with liposomes.

The N-terminal signal sequence of glucitol permease of Escherichia coli (Gut22: MIETITPGAVWFIGLFQKGGEC) and its analog (Gut22Ana: MIETITHGAEWFIGLFQKGGEC) were synthesized. The analog had a Pro residue substituted for the His at the 7th position of Gut22 and a Val residue substituted for the Glu at the 10th position. Previous studies indicated that due to its structural rigidity, the interaction of Gut22Ana with lipid bilayer was much weaker than that of Gut22 (Wang, Q.D., Cui, D.F. and Lin, Q.S. (1996) Science in China (Series C) 39, 395-405). To further probe the location of the tryptophan residues of the peptides in lipid bilayer, the membrane penetration depth of the tryptophan residue of Gut22 was measured using spin-labeled phospholipids, and fluorescence quenching of the peptides by iodide and acrylamide in the presence and absence of phosphatidylserine/phosphatidylcholine liposomes were also studied. Fluorescent labeling of the peptides enabled the study of their association with membrane by fluorospectrophotometry. In the presence of liposomes, the peptides were protected from reaction with chymotrypsin, indicating that the peptide incorporated into the membrane. However, dithionite, which acts external to the membrane, reacted with the peptide, showing that the peptides did not translocate across lipid bilayer spontaneously.

Acrylamide↗
PubMed1991

Inhibition of O2-. generating oxidase of neutrophils by iodonium biphenyl in a cell free system: effect of the redox state of the oxidase complex.

The conditions of inhibition of neutrophil O2-. generating oxidase by iodonium biphenyl (IBP) were studied. In a cell free system of oxidase activation consisting of neutrophil membranes and cytosol, GTP-gamma-S, Mg2+ and arachidonic acid, the inhibitory effect of IBP depended on the redox conditions of the medium. Inhibition was observed when the medium was supplemented with dithionite or NADPH. When the cell free system was incubated with IBP in the absence of reducing agents, full oxidase activity was recovered after removal of free IBP by gel filtration. Bovine neutrophil membranes, but not cytosol, contained component(s) sensitive to IBP. Upon treatment of neutrophil membranes by IBP followed by reduction, the spectrum of reduced cytochrome b558 was modified, suggesting that cytochrome b558 is a target site for IBP.

Animals↗
PubMed1992

Interaction of ubiquinone and vitamin K3 with mitochondrial succinate-ubiquinone oxidoreductase.

Two new methods have been devised for measuring fumarate reduction by beef heart succinate-ubiquinone oxidoreductase with quinols as electron donors. In one assay the quinone is maintained in the reduced state by coupling fumarate reduction with the DT-diaphorase reaction, in the other assay by the presence of excess dithionite. The advantages of these methods are discussed, along with preliminary characterization of the quinol-protein interaction.

Animals↗
PubMed1982

Continuous flow-resonance Raman spectroscopy of an intermediate redox state of cytochrome C.

An intermediate redox state of cytochrome c at alkaline pH, generated upon rapid reduction by sodium dithionite, has been observed by resonance Raman (RR) spectroscopy in combination with the continuous flow technique. The RR spectrum of the intermediate state is reported for excitation both in the (alpha, beta) and the Soret optical absorption band. The spectra of the intermediate state are more like those of the stable reduced form than those of the stable oxidized form. For excitation of 514.5 nm, the most prominent indication of an intermediate state is the wave-number shift of one RR band from 1,562 cm-1 in the stable oxidized state through 1,535 cm-1 in the intermediate state to 1,544 cm-1 in the stable reduced state. For excitation at 413.1 nm, a band, present at 1,542 cm-1 in the stable reduced state but not present in the stable oxidized state, is absent in the intermediate state. We interpret the intermediate species as the state where the heme iron is reduced but the protein remains in the conformation of the oxidized state, with methionine-80 displaced as sixth ligand to the heme iron, before relaxing to the conformation of the stable reduced state, with methionine-80 returned as sixth ligand.

Cytochrome c Group↗
PubMed1985

Naturally crystalline hemoglobin of the nematode Mermis nigrescens. An in situ microspectrophotometric study of chemical properties and dichroism.

A dichroic microspectrophotometer was used to measure isotropic and dichroic absorbance spectra of this unique cytoplasmic hemoglobin and its derivatives. A perfusion slide enabled changing the media bathing the Mermis head. The native spectrum, which has an exceptionally low alpha-band extinction, was shown to be entirely due to oxyhemoglobin. The CO-hemoglobin spectrum is more typical, however, the alpha- and beta-bands are unusually closely spaced. A ferric hemochrome was formed on oxidation with ferricyanide or hydroxylamine and was readily converted to ferric hemoglobin cyanide on adding cyanide. Aquoferric hemoglobin and ferric hemoglobin fluoride were not easily formed. Deoxyhemoglobin, identified by its typical absorption spectrum, was formed only under the extremely low O2 pressures attainable in the presence of dithionite. A glucose oxidase, catalase solution deoxygenated hemoglobin in human erythrocytes but not in adjacent Mermis preparations. The affinity for O2 is much greater than for CO. Also, spectral evidence points to an oxyheme environment that is different than in vertebrate hemoglobin and myoglobin. The polarization ratio (PR) magnitude and the PR spectrum were unaffected by perfusion with high refractive index solvents; therefore, form dichroism due to the rodlike crystals is negligible. Maximum extinction is approximately perpendicular to the long axis of the microscopic crystals, which are oriented parallel to the body axis within the hypodermal cells. The PR spectra of the hemoglobin derivatives strongly resemble the corresponding spectra previously reported of single crystals made of horse hemoglobin, whale myoglobin, or Aplysia myoglobin and change appropriately when the ligand is changed. This confirms that the intracellular crystals of Mermis are of oxyhemoglobin.

Animals↗
PubMed1999

The Saccharomyces cerevisiae succinate-ubiquinone reductase contains a stoichiometric amount of cytochrome b562.

The Saccharomyces cerevisiae succinate-ubiquinone reductase or succinate dehydrogenase (SDH) is a tetramer of non-equivalent subunits encoded by the SDH1, SDH2, SDH3, and SDH4 genes. In most organisms, SDH contains one or two endogenous b-type hemes. However, it is widely believed that the yeast SDH does not contain heme. In this report, we demonstrate the presence of a stoichiometric amount of cytochrome b562 in the yeast SDH. The cytochrome is detected as a peak present in fumarate-oxidized, dithionite-reduced mitochondria. The peak is centered at 562 nm and is present at a heme:covalent FAD molar ratio of 0.92+/-0.11. The cytochrome is not detectable in mitochondria isolated from SDH3 and SDH4 deletion strains. These observations strongly support our conclusion that cytochrome b562 is a component of the yeast SDH.

Animals↗
PubMed1999

Anoxic function for the Escherichia coli flavohaemoglobin (Hmp): reversible binding of nitric oxide and reduction to nitrous oxide.

The flavohaemoglobin Hmp of Escherichia coli is inducible by nitric oxide (NO) and provides protection both aerobically and anaerobically from inhibition of growth by NO and agents that cause nitrosative stress. Here we report rapid kinetic studies of NO binding to Fe(III) Hmp with a second order rate constant of 7.5 x 10(5) M(-1) s(-1) to generate a nitrosyl adduct that was stable anoxically but decayed in the presence of air to reform the Fe(III) protein. NO displaced CO bound to dithionite-reduced Hmp but, remarkably, CO recombined after only 2 s at room temperature indicative of NO reduction and dissociation from the haem. Addition of NO to anoxic NADH-reduced Hmp also generated a nitrosyl species which persisted while NADH was oxidised. These results are consistent with direct demonstration by membrane-inlet mass spectrometry of NO consumption and nitrous oxide production during anoxic incubation of NADH-reduced Hmp. The results demonstrate a new mechanism by which Hmp may eliminate NO under anoxic growth conditions.

Anaerobiosis↗
PubMed1979

Cytochrome b is present in neutrophils from patients with chronic granulomatous disease.

Analysis of dithionite difference spectra demonstrated that cytochrome b was present in neutrophil homogenates from a 17-year-old girl and her 25-year-old brother who had the autosomal recessive form of chronic granulomatous disease, and from an 18-year-old boy with the X-linked form of chronic granulomatous disease. These results indicate that the postulated importance of cytochrome b in the oxygen burst during phagocytosis is questionable.

Adolescent↗
PubMed2000

EPR evidence of two structurally different diferric sites in Mycobacterium tuberculosis R2-2 ribonucleotide reductase protein.

Mixed-valent species were generated in the diiron site of active (with tyrosyl free radical) and met (without radical) forms of protein R2-2 in a class Ib ribonucleotide reductase from Mycobacterium tuberculosis by low temperature reduction (gamma-irradiation) at 77 K. The primary mixed-valent EPR signal is a mixture of two components with axial symmetry and g(av) < 2.0, observable at temperatures up to 77 K, and assigned to antiferromagnetically coupled high spin ferric/ferrous sites. The two components in the primary EPR signal can be explained by the existence of two structurally distinct mu-oxo-bridged diferric centers, possibly related to structural heterogeneity around the iron site, and/or different properties of the two polypeptide chains in the homodimeric protein after the radical reconstitution reaction. Annealing of the irradiated R2-2 samples to 143 K transforms the primary EPR signal into a rhombic spectrum characterized by g(av) < 1.8 and observable only below 25 K. This spectrum is assigned to a partially relaxed form with a mu-hydroxo-bridge. Further annealing at 228 K produces a new complex rhombic EPR spectrum composed of at least two components. An identical EPR spectrum was observed and found to be stable upon chemical reduction of Mycobacterium tuberculosis RNR R2-2 at 293 K by dithionite.

Dithionite↗
PubMed1981

Studies on heme d1 extracted from Pseudomonas aeruginosa nitrite reductase.

Heme d1 has been extracted from Pseudomonas nitrite reductase. Imidazole, cyanide, and chloride-ferroheme, and CO, NO, cyanide, imidazole, and pyridine-ferroheme complexes have been prepared for study by UV/vis spectroscopy, and in some cass by epr and low-temperature mcd as well. Iron determinations have been carried out to assess extinction coefficients. Absorption spectra were used to monitor the transition of chloride-ferriheme d1 to an alkaline form of ferriheme d1 and a pka of 6.5 was determined for the process. The epr spectrum of chloride-ferriheme possessed the characteristic g = 6 signal of high spin (S = 5/2) iron, but the alkaline-ferriheme form gave no detectable epr signals. Electron paramagnetic resonance spectra were also obtained for cyanide and imidazole-ferriheme d1 and for NO-ferroheme d1. The imidazole complex gave signals that were very weak in comparison with the cyanide complex, but mcd measurements of imidazole-ferriheme d1 were consistent with it being a low-spin (S = 1/2) system. The epr signals of NO-ferroheme d1 were similar to those of the corresponding holo-enzyme complex. Reduction of alkaline-ferriheme d1 was found to be affected by the presence of oxygen, but under N2 give the same result with ascorbate and dithionite. Autoreduction of alkaline-ferriheme d1 was observed when placed under CO, and NO, atmospheres, or when treated with pyridine.

Carbon Monoxide↗
PubMed2003

Dihydrolipoic acid inhibits 15-lipoxygenase-dependent lipid peroxidation.

The potential antioxidant effects of the hydrophobic therapeutic agent lipoic acid (LA) and of its reduced form dihydrolipoic acid (DHLA) on the peroxidation of either linoleic acid or human non-HDL fraction catalyzed by soybean 15-lipoxygenase (SLO) and rabbit reticulocyte 15-lipoxygenase (RR15-LOX) were investigated. DHLA, but not LA, did inhibit SLO-dependent lipid peroxidation, showing an IC(50) of 15 microM with linoleic acid and 5 microM with the non-HDL fraction. In specific experiments performed with linoleic acid, inhibition of SLO activity by DHLA was irreversible and of a complete, noncompetitive type. In comparison with DHLA, the well-known lipoxygenase inhibitor nordihydroguaiaretic acid and the nonspecific iron reductant sodium dithionite inhibited SLO-dependent linoleic acid peroxidation with an IC(50) of 4 and 100 microM, respectively, while the hydrophilic thiol N-acetylcysteine, albeit possessing iron-reducing and radical-scavenging properties, was ineffective. Remarkably, DHLA, but not LA, was also able to inhibit the peroxidation of linoleic acid and of the non-HDL fraction catalyzed by RR15-LOX with an IC(50) of, respectively, 10 and 5 microM. Finally, DHLA, but once again not LA, could readily reduce simple ferric ions and scavenge efficiently the stable free radical 1,1-diphenyl-2-pycrylhydrazyl in ethanol; DHLA was considerably less effective against 2,2'-azobis(2-amidinopropane) dihydrochloride-mediated, peroxyl radical-induced non-HDL peroxidation, showing an IC(50) of 850 microM. Thus, DHLA, at therapeutically relevant concentrations, can counteract 15-lipoxygenase-dependent lipid peroxidation; this antioxidant effect may stem primarily from reduction of the active ferric 15-lipoxygenase form to the inactive ferrous state after DHLA-enzyme hydrophobic interaction and, possibly, from scavenging of fatty acid peroxyl radicals formed during lipoperoxidative processes. Inhibition of 15-lipoxygenase oxidative activity by DHLA could occur in the clinical setting, eventually resulting in specific antioxidant and antiatherogenic effects.

Acetylcysteine↗
PubMed2000

Unequivocal determination of metal atom oxidation state in naked heme proteins: Fe(III)myoglobin, Fe(III)cytochrome c, Fe(III)cytochrome b5, and Fe(III)cytochrome b5 L47R.

Unambiguous determination of metal atom oxidation state in an intact metalloprotein is achieved by matching experimental (electrospray ionization 9.4 tesla Fourier transform ion cyclotron resonance) and theoretical isotopic abundance mass distributions for one or more holoprotein charge states. The ion atom oxidation state is determined unequivocally as Fe(III) for each of four gas-phase unhydrated heme proteins electrosprayed from H2O: myoglobin, cytochrome c, cytochrome b5, and cytochrome b5 L47R (i.e., the solution-phase oxidation state is conserved following electrospray to produce gas-phase ions). However, the same Fe(III) oxidation state in all four heme proteins is observed after prior reduction by sodium dithionite to produce Fe(II) heme proteins in solution: thus proving that oxygen was present during the electrospray process. Those results bear directly on the issue of similarity (or lack thereof) of solution-phase and gas-phase protein conformations. Finally, infrared multiphoton irradiation of the gas-phase Fe(III)holoproteins releases Fe(III)heme from each of the noncovalently bound Fe(III)heme proteins (myoglobin, cytochrome b5 and cytochrome b5 L47R), but yields Fe(II)heme from the covalently bound heme in cytochrome c.

Animals↗
PubMed2002

Inhibitors alter the spectrum and redox properties of monoamine oxidase A.

Monoamine oxidase A (MAO A) catalyses the oxidation of both neurotransmitter and ingested amines. The mechanism of catalysis involves the covalently bound FAD cofactor. Although substrates and inhibitors alter the thermodynamic and kinetic properties of the flavin, how the ligands interact with the flavin is unknown. This work characterises the spectral changes that occur on inhibitor binding to MAO A and examines how the binding influences the flavin. The inhibitors, D-amphetamine, harmine, tetrindole, and befloxatone all induce similar (but not identical) changes in the spectrum of MAO A, consistent with stacking of inhibitor with the flavin in the active site. D-Amphetamine, harmine, and tetrindole stabilise the semiquinone form of FAD during reduction of MAO A by dithionite and no further reduction of these inhibitor-MAO A complexes has been observed. In contrast, semiquinone is never observed during reduction of the befloxatone-MAO A complex. Instead, partial reduction directly to the FADH(2) form occurs extremely slowly. Thus, inhibitor binding has a strong, structure-dependent influence on the environment of the flavin that alters its electronic properties.

Carbazoles↗
PubMed1995

Flow cytometric analysis of transmembrane phospholipid movement in bull sperm.

Fluorescent phospholipids are useful to investigate phospholipid dynamics in biological membranes. We used flow cytometry to investigate transbilayer phospholipid movement in live sperm cells. Acyl-labeled N-4-nitrobenzo-2-oxa-1,3-diazole (NBD) -phosphatidylcholine (-PC), -phosphatidylethanolamine (-PE), or -phosphatidylserine (-PS) were incorporated into sperm cells, and the transbilayer location was determined by extraction of probe from cell with excess bovine serum albumin (BSA) or by chemical destruction of probe by sodium dithionite. Using these methods, we have measured the head group specific outer leaflet to inner leaflet movement (flip) of the aminophospholipids NBD-PS and NBD-PE. The fluorescent phospholipids moved inward across the plasma membrane with half-times of 1.8, 2.5, and 11.2 min, for NBD-PS, NBD-PE, and NBD-PC and reached apparent equilibrium levels of 88%, 94%, and 32% inside, respectively. The inward movement of NBD-PE was inhibited by sulfhydryl reagents, elevated intracellular Ca2+, and depletion of cellular ATP. Analysis of the kinetics of NBD-PE and -PS extraction by BSA allows determination of the rates for outward movement (flop) across the plasma membrane. Half-times for flop were 4.7 and 4.5 min for NBD-PS and -PE, respectively. Based on these measurements, a simple model of NBD-phospholipid equilibria was developed and fit to the kinetic data. Computer-generated fits reflected major features of the experimental data and provide a potential tool for predicting the dynamics of endogenous lipids.

4-Chloro-7-nitrobenzofurazan↗
PubMed1995

Interconversion of fast and slow forms of cytochrome bo from Escherichia coli.

The fully oxidized fast form of cytochrome bo from Escherichia coli is shown to convert spontaneously to a slow form when stored at -20 degrees C in 50 mM potassium borate, pH 8.5, containing 0.5 mM potassium EDTA. Evidence for the conversion, and that the form produced is analogous to the slow form of bovine heart cytochrome c oxidase, comes from (a) decreases in the extents of fast (k = 1-2 x 10(3) M-1 s-1) H2O2 binding and fast (k = 20-30 M-1 s-1) cyanide binding; (b) changes in the optical spectrum that are like those induced by formate, i.e., a blue shift in the Soret absorption band, loss of absorbance in the alpha and beta bands, and a red shift in the "630 nm" charge-transfer band; (c) changes in the EPR spectrum that are like those induced by formate, i.e., disappearance of signals at g = 8.6 and g = 3.71, and appearance of signals at g approximately 13, g = 3.14, and g = 2.58; and (d) appearance of a slow phase of reduction of heme o by dithionite. The mutant enzyme E286Q also converts to a slow form under the same conditions, as shown by (a) a decrease in the extent of fast H2O2 binding; (b) changes in the optical spectrum like those seen with wild-type enzyme; and (c) changes in the EPR spectrum that are like those induced by formate, i.e., disappearance of signals at g = 7.3 and g = 3.6 and appearance of signals at g approximately 13, g = 3.18, and g = 2.59.(ABSTRACT TRUNCATED AT 250 WORDS)

Borates↗
PubMed2000

The FMN-binding domain of cytochrome P450BM-3: resolution, reconstitution, and flavin analogue substitution.

Cytochrome P450BM-3 is a self-sufficient bacterial protein containing three naturally fused domains which bind either heme, FMN, or FAD. Resolution of protein and FMN from the isolated FMN-containing domain of cytochrome P450Betamicro-3 was accomplished using trichloroacetic acid. The apoprotein thus prepared was shown to rebind FMN to regenerate the original holoprotein as indicated by both spectroscopy and activity measurements. To better understand how the protein/flavin interaction might contribute to reactivity, the association process was studied in detail. Fluorescence quenching was used to measure a dissociation constant of the flavin-protein complex of 31 nM, comparable to FMN-containing proteins of similar reactivity and higher than that of flavodoxins. Stopped-flow kinetics were performed, and a multistep binding process was indicated, with an initial k(on) value of 1.72 x 10(5) M(-)(1) s(-)(1). Preparation of the apoprotein allowed substitution of flavin analogues for the native FMN cofactor using 8-chloro-FMN and 8-amino-FMN. Both were found to bind efficiently to the protein with only minor variations in affinity. Reductive titrations established that, as in the native FMN-containing FMN-binding domain, the 8-amino-FMN-substituted domain does not produce a stable one-electron-reduced species during titration with sodium dithionite. The 8-chloro-FMN-substituted domain, however, had sufficiently altered redox properties to form a stable red anionic semiquinone. The 8-chloro-FMN-substituted FMN-binding domain was shown in reconstituted systems to retain most of the cytochrome c reductase activity of the native domain but only a very small amount of palmitic acid hydroxylase activity. The 8-amino-FMN-substituted FMN-binding domain showed no palmitic acid hydroxylase activity and only 30% of the native cytochrome c reductase activity, demonstrating the importance of thermodynamics to the mechanism of this protein.

Anaerobiosis↗
PubMed1995

Continuous analysis of the mechanism of activated transbilayer lipid movement in platelets.

Dithionite reduction of fluorescent (NBD) phospholipids was used as the basis of a continuous assay of transbilayer lipid movement to the cell surface during platelet activation. This assay reveals that virtually all previously internalized phosphatidylserine passes through the external leaflet of the membrane within 90 s after activation with Ca2+ and ionophore or with thrombin and thapsigargin. We demonstrate that this lipid scrambling is reversible, bidirectional, and insensitive to the lipid headgroup. Prolonged activation gradually results in inactivation of the scramblase. The assay also reveals that activation of the scrambling activity is sensitive to the sulfhydryl reagent pyridyldithioethylamine, suggesting the involvement of a protein in the process of activated transbilayer lipid scrambling.

4-Chloro-7-nitrobenzofurazan↗