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PubMed1983

Oxidation of 2-thioflavins by peroxides. Formation of flavin 2-S-oxides.

The reaction of 2-thioriboflavin (sulfur replacing the oxygen substituent at position 2 alpha) with hydrogen peroxide at pH approximately 10 leads to a blue flavin (lambda max = 565 nm) which was purified in stable, homogeneous form. Titrations of 2-thioflavins with m-chloroperoxybenzoic acid also yield the same blue flavins with consumption of 1 eq of peracid. Anaerobic reduction of the blue flavin by sodium dithionite requires 4e- eq, and leads to formation of 1,5-dihydro-2-thioflavin. Oxidation of the latter with O2 restores the original 2-thioflavin. pH titration of the blue flavin shows two pKa values of 2.4 and 6.6, with no apparent ionization in the pH range 8-11. These results suggest that the blue flavin is a flavin 2-S-oxide. The visible absorption spectra of flavin 2-S-oxides show a pronounced dependence on solvent polarity. This property suggests that these flavin analogs may be useful hydrophilic/hydrophobic probes of flavoprotein active sites. Flavin 2-S-oxides can be oxidized further to the 2-sulfinate and 2-sulfonate analogs, some properties of which are described.

Chromatography, High Pressure Liquid↗
PubMed1982

Charge transfer complexes between pteridine substrates and the active center molybdenum of xanthine oxidase.

Anaerobic addition of lumazine (2,4-dihydroxypteridine) to xanthine oxidase leads to a rapid bleaching of the enzyme with concomitant formation of a long wave-length species with maximum absorbance at 650 nm. At the conclusion of the reaction there is a net increase in absorbance at wavelengths greater than 550 nm. A spectrally similar species is obtained when violapterin is added to dithionite-reduced enzyme. This long wavelength absorbance is not accompanied by an EPR signal characteristic of flavin neutral radical and it is produced equally well in flavin-free xanthine oxidase. Its production is eliminated by the inhibitors, cyanide and allopurinol, which react at the molybdenum center. We conclude that this new species is a charge transfer complex between Mo(IV) and the product of the reaction, violapterin (2,4,7-trihydroxypteridine). A similar, though less intense, absorbance at 650 nm is also obtained when lumazine is bound to the reduced enzyme. These Mo(IV)-pteridine charge transfer compounds are optically active and exhibit intense circular dichroism centered at 630 nm.

Anaerobiosis↗
PubMed1981

Steady state kinetics of erythrocyte anion exchange. Evidence for site-site interactions.

Steady state velocities for the rate of exchange of extracellular dithionite for intracellular sulfate were measured in the presence of single and pairs of cis inhibitors. The purpose of these kinetic studies was to test the current two site model used to explain the nonhyperbolic transport kinetics characteristic of the erythrocyte anion exchange system. This model suggests the existence of a transport site and an external, pure noncompetitive inhibitory modifier site, both of which are thought to exist on each subunit of the band 3 protein dimer. Our results show negative cooperative transport curves when the velocity is plotted versus the external anion concentration at constant trans anion concentration. Reversible inhibition by SITS (4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonate) caused the degree of negative cooperativity to change nonlinearly as a function of SITS concentration. This nonlinear dependence was such that SITS actually increased the calculated degree of negative cooperativity and then decreased it suggesting site-site interactions. Multiple inhibition studies confirm the existence of site-site interactions by showing that SITS and acetate are nonmutually exclusive inhibitors with negative interactions or mutually hindered inhibitor binding. These results are not consistent with the transport site-modifier site kinetic model in its original form since that model proposes only one pathway for transport with no effect of one inhibitor on the binding of the other. Possible kinetic models are discussed in an attempt to explain the present results and to relate them to what is currently known about the structure of band 3 protein.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1980

Identification of an endogenous electron donor for biohydrogenation as alpha-tocopherolquinol.

Four fluorescent compounds present in solvent extracts of Butyrivibrio fibrisolvens could serve as electron donors for the biohydrogenation of cis-9,trans-11-octadecadienoate in the presence of dithionite, which was itself inactive. One of the compounds was identified as alpha-tocopherolquinol and another as alpha-tocopherolquinone. A partially purified soluble enzyme preparation from B. fibrisolvens catalyzed the reduction of alpha-tocopherolquinone to alpha-tocopherolquinol in the presence of NADH with a stoichiometry of 1:1. The ratio of alpha-tocopherolquinone produced to fatty acid reduced was 2:1 when the tocopherol derivatives were extracted aerobically. When the extraction was carried out anaerobically, the ratio was 1. It is suggested that the oxidation of 2 molecules of alpha-tocopherolquinol, each to the semiquinone, provides the electrons required for the reduction of the cis-bond of the conjugated dienoic fatty acid. Although alpha-tocopherol, phylloquinol, and reduced menadione are inactive, ubiquinol-4, ubiquinol-10, and trimethylhydroquinone show about one-half the activity of alpha-tocopherolquinol. Plastoquinol and trimethylphytylbenzoquinol are as active as alpha-tocopherolquinol.

Anaerobiosis↗
PubMed1980

[Determination of serum iron; a comparison of two methods: Teepol/dithionite/bathophenanthroline versus guanidine/ascorbic acid/Ferrozine (author's transl)].

Two spectrophotometric methods for the assay of serum iron without deproteinisation are compared: 1. Liberation of iron by a detergent (Teepol SHELL), reduction by dithionite, chelation by bathopehanthroline disulfonate; 2. Liberation of iron by 6 mol/l guanidine, reduction by ascorbic acid, chelation by 3-(2-pyridyl)-5,6-bis-(4-phenyl sulfonic acid)-1,2,4-triaxine (Ferrozine).

Ascorbic Acid↗
PubMed1981

Analysis for paraquat by second- and fourth-derivative spectroscopy.

We describe simple, precise methods, based on second and fourth-derivative spectroscopy, of analysis for paraquat in serum, plasma, or dialysis fluid. Plasma paraquat, after ion-pair extraction and dithionite reduction, yields a sharp, stable absorption peak at 396 nm. Derivative spectroscopy confers enhanced sensitivity (detection limit, 5 microgram/L) and improved rejection of spectroscopic interference by diquat, hemolysis, bilirubin, or lipemia. Derivative peak amplitudes are linearly related to paraquat dichloride concentration from 10 to 1000 microgram/L in plasma, and the methods are precise (CV 2.7-5.2% for 50 microgram/L concentration) and accurate. Results of derivative assays for 18 cases of paraquat poisoning correlate well with those by radioimmunoassay (r = 0.999). Derivative spectroscopy enables monitoring of paraquat concentrations in the plasma of poisoned patients during treatment by hemoperfusion or hemodialysis. A rapid assay developed for dialysis fluid (detection limit, 2 microgram/L) permits the efficacy of hemodialysis to be routinely assessed.

Diquat↗
PubMed1976

Requirement of an essential thiol group and ferric iron for the activity of the progesterone-induced porcine uterine purple phosphatase.

The progesterone-induced purple phosphatase isolated from the uterine flushings of pigs is activated by a variety of reagents that cleave disulfide bonds, including 2-mercaptoethanol, dithiothreitol, L-ascorbate, L-cysteine, sulfite, and cyanide. It is inhibited by various mercurials, iodoacetamide, O-iodosobenzoate, and hydrogen peroxide. Thiols increase the specific phosphatase activity from 25 to about 300 units per mg of enzyme. This activation is accompanied by a shift in the extinction maximum to higher energy to yield a protein with a pink coloration. Following maximum activation there is a gradual decrease in enzyme activity and protein color which is accompanied by loss of ferrous iron from the protein. Sodium dithionite at 10 mM or higher causes an immediate inhibition of phosphatase activity and bleaching of color, and can be used to prepare the iron-free apoprotein. The latter can be partially reactivated by Fe3+ salts but not by Fe2+. The Fe3+ restores the pink form of the enzyme with a specific activity of about 200 units/mg of protein. Cu2+ also causes some reactivation, but other metal ions were ineffective. ESR studies showed that the pink form of phosphatase contains approximately 1 atom of high spin ferric iron per molecule. It is concluded that the phosphatase requires a free thiol and Fe3+ for activity. Reduction of the iron leads to complete loss of both color and enzyme activity. The color change from purple to pink represents disulfide reduction and is not due to reduction of iron.

Acid Phosphatase↗
PubMed1993

Effect of hypoxia on the intracellular chloride activity of cultured glomus cells of the rat carotid body.

Clusters of rat carotid body glomus cells were cultured for 1 to 12 days. Preparations were exposed to a control PO2 of 134.9 +/- 1.1 torr (mean +/- SE), and an external chloride activity, ao(Cl), of 105.6 +/- 2 mM. Thirty-six cells had a resting potential (Em) of -25.2 +/- 0.9 mV. The intracellular chloride activity, ai(Cl), was 32.8 +/- 1.1 mM, and the calculated chloride equilibrium potential (ECl) was -30.9 +/- 0.9 mV. ECl was more negative than Em, indicating that Cl- ions are not passively distributed. Hypoxia (5.4 +/- 0.8 torr), induced by Na-dithionite (Na2S2O4) 1.25 mM, elicited cell depolarization, increased ai(Cl) and a less negative ECl in about 80% of the cells. Fourteen per cent of the cells showed opposite effects. It is hypothesized that the increased ai(Cl) occurs because an outward-directed chloride pump is blocked by hypoxia. This effect is aided by depolarization of the cell. Decreased ai(Cl) may be due to cell hyperpolarization.

Animals↗
PubMed1979

The rate of oxygen uptake by human red blood cells.

Oxygen uptake into intact and reconstituted human red blood cells was measured using dual wavelength, stopped flow techniques. The rate of oxygen uptake by human erythrocytes is roughly 40 times slower (t 1/2 congruent to 80 ms at 0.125 mM O2, 25 degrees C) than the corresponding rate of oxygen combination with free hemoglobin. Oxygen transport through the red cell cytoplasm accounts for part of this difference and predicts a half-time of uptake of about 15 ms, which is still 5 times smaller than that observed experimentally. Further limitation of uptake appears to be due to the presence of unstirred layers of solvent adjacent to the red cell surface. Very rapidly after mixing, these layers form and become depleted of O2 due to uptake by the cells. This requires that the bulk of the oxygen molecules must diffuse over rather large distances, 1.0 to 5.0 micrometer, before they can penetrate the erythrocytes. A mathematical model was developed to take into account diffusion through an unstirred solvent layer which increases in thickness with time. This scheme can account quantitatively both for the dependence of the apparent rate of uptake on O2 concentration and for the shape of the observed time courses. In addition, the diffusion parameters which were developed for the O2 reaction can also be used to describe quantitatively the rates and time courses of CO and ethyl isocyanide uptake and the rates and time courses of O2 release from cells in the presence of sodium dithionite. Finally, the parameters used to describe the stopped flow results can also be used to simulate quantitatively O2 uptake time courses obtained from previous studies with thin films of red cells (Sinha, A. K. (1969) Ph.D. dissertation, University of California, San Francisco; Thews, G. (1959) Arch. Gesamte Physiol. Mens. Tiere (Pflufgers) 268, 308-317).

Diffusion↗
PubMed1994

Purification and analysis of a flavoprotein functional as NADH oxidase from Amphibacillus xylanus overexpressed in Escherichia coli.

The gene encoding the Amphibacillus xylanus flavoprotein has been cloned into pTTQ18 and overexpressed in Escherichia coli. The recombinant enzyme has been purified to homogeneity yielding 15 mg of pure enzyme/liter of cell culture. Recombinant flavoprotein is fully active and has an absorption spectrum identical to that of the enzyme purified from A. xylanus. The N-terminal sequence analysis and analytical gel filtration data confirm the structural identity of recombinant and A. xylanus enzymes. The Km value for oxygen and the Km value for NADH are 1.7 mM and 33.3 microM, respectively. In the presence of free additional FAD, however, the Km value for oxygen decrease dramatically. The NADH oxidase activity is accelerated markedly in the presence of additional FAD. The intracellular free FAD concentration of A. xylanus is calculated about 13 microM. This FAD concentration would be enough to accelerate the NADH oxidase activity of flavoprotein in cells of A. xylanus. Two-electron reduction of the enzyme FAD by the strong reductant dithionite occurs during the total uptake of 6 electrons. Such behavior usually indicates the presence of non-flavin redox centers. The high degree of homology between this enzyme and alkyl hydroperoxide reductase F52a protein and thioredoxin reductase suggests that these centers are the redox-active disulfide adjacent to the FAD and another disulfide, which is able to slowly interchange with the redox-active disulfide. The presence of two disulfides has been demonstrated.

Amino Acid Sequence↗
PubMed1975

Partial characterization of hepatic micorsomes from tupaia glis.

The dithionite difference spectrum of Tupaia microsomal cytochrome P-450-CO complex has an absorbance maximum at 449 nm, rather than at 450 nm as found for rat, rabbit, mouse, guinea pig, and human microsomes. N-octylamine difference spectroscopy showed the ratio of high- and low-spin forms of cytochrome P-450 to be different from that of the rat, indicating that the 449 nm absorption maximum might be due to a high concentration of the high-spin form, cytochrome P-448. The Tupaia microsomes demonstrated greater aryl hydrocarbon activity than microsomes prepared from male rats. These preliminary results suggest that Tupaia microsomes contain a modified terminal oxygenase which is responsible for the observed rapid metabolism of benzo[alpha]pyrene.

Animals↗
PubMed1993

Production of recombinant cytochrome b558 allows reconstitution of the phagocyte NADPH oxidase solely from recombinant proteins.

Phagocytic white blood cells contain a multicomponent oxidase that generates microbicidal products by catalyzing electron transfer from NADPH to molecular oxygen. Activation of this oxidase requires interactions of a unique membrane flavocytochrome with the cytosolic proteins p47phox, p67phox, and p21Rac. This flavocytochrome, designated cytochrome b558, is a heteromer comprising a 22-kDa alpha-subunit (p22phox) and a glycosylated approximately 91-kDa beta-subunit (gp91phox). Cytochrome b558 was expressed in Sf9 insect cells coinfected with recombinant baculoviruses carrying cDNAs for p22phox and gp91phox. Membranes of these cells contained a b-type cytochrome with a dithionite-reduced minus oxidized difference spectrum similar to that of neutrophil cytochrome b558. The recombinant cytochrome b558 beta-subunit was heterogeneously N-glycosylated as demonstrated by its susceptibility to cleavage with endoglycosidases F and H. In contrast to the neutrophil cytochrome b558, a portion of the N-linked oligosaccharide was of the high mannose type. Recombinant cytochrome b558 supported superoxide production in a cell-free assay containing recombinant p47phox, p67phox, and p21Rac. The enzymatic turnover of the partially purified recombinant cytochrome b558 and neutrophil cytochrome b558 were similar (approximately 100-160 mol of superoxide generated/s/mol of cytochrome heme, range of two experiments) and the native and recombinant cytochromes showed similar requirements for NADPH and exogenous FAD. These studies represent the first reconstitution of the NADPH oxidase solely from recombinant proteins and define a model system to explore the structure and function of cytochrome b558.

Amino Acid Sequence↗
PubMed1977

Preparation and properties of immobilized rubredoxin.

Rubredoxin, one of the three protein components of the epoxidation/hydroxylation system of Pseudomonas oleovorans was immobilized by attachment to CNBr-activated agarose (Sepharose 4B). Since this represents the first reported example of the preparation of a water-insoluble derivative of an enzyme of this type, the electron transfer and physical properties of the conjugate were examined in order to allow comparison with those of the soluble enzyme. Immobilized rubredoxin exhibits all of the major spectral properties of the soluble enzyme above 300 nm, but some distortion in the 280 nm abosrbance band was observed. The immobilized enzyme accepts electrons from dithionite or form NADPH in the presence of spinach ferredoxin-NADP reductase, and upon reduction the visible absorbance is bleached. Immobilized rubredoxin mediates the reduction of cytochrome c in the presence of NADPH and spinach reductase, although it is less efficient in this role than soluble rubredoxin. The oxidation-reduction potential of immobilized rubredoxin was determined and found to be similar to that of the soluble enzyme. In the presence of 2.5 m guanidine HCL, the immobilized enzyme is considerably more stable than soluble rubredoxin toward denaturation. After anaerobic reduction, iron was readily removed from immobilized rubredoxin by washing in 0.5 m Tris base, PH 9.5 containing 0.07 M mercaptoethanol, and the resulting immobilized apoenzyme could then be reconstituted to give back a conjugate with the original iron content, as judged from its absorbance at 497 NM. Reptition of the entire reduction-dissociation-reconstitution cycle gave the same results as were obtained after the initial reconstitution.

Anaerobiosis↗
PubMed1977

Reductive titration of cytochrome P-450 in rat liver microsomes.

Microsomal cytochromes b5 and P-450 of rat liver have been titrated with standardized sodium dithionite solution with a newly developed titrating apparatus that allows spectrophotometric monitoring of the reduction process while strictly O2-free conditions are maintained throughout the procedure. Cytochrome b5 and other electron acceptors in the microsomal preparation were saturated with reducing equivalents prior to addition of carbon monoxide to the system. Continued titration in the presence of CO revealed that 1 electron equivalent was required for the formation of P-450(Fe2+)-CO. These results are in agreement with previous findings of 1 electron equivalence for cytochrome P-450 of adrenocortical mitochondria and P-450CAM.

Anaerobiosis↗
PubMed1999

Polypyrrole-heparin composites as stimulus-responsive substrates for endothelial cell growth.

Heparin is a potent anticoagulant which can be immobilized on biomaterial surfaces to increase their hemocompatability. In the present work, we have electrochemically synthesized composites comprising heparin and the electrically conducting polymer polypyrrole. The incorporation and exposure of heparin were controlled by varying key conditions of polymer synthesis (i.e., applied current and synthesis time). The resulting composite polymers were electroactive after synthesis and the amount of heparin exposed in the polymer could be increased (up to threefold) by switching the polymers from their oxidized to reduced states. Polymer reduction was achieved by either application of negative potentials (-0.4 to -0.7 V for 90 s) or exposure to aqueous reductant (0.1M sodium dithionite for 30 min). Heparin-polypyrrole composites remained stable after autoclaving, displaying no significant loss of electroactivity, and had a shelf life of at least 2 years postautoclaving. Finally, the composites were found to be excellent substrates for the growth of human endothelial cells.

Biocompatible Materials↗
PubMed2002

Synthesis and membrane binding properties of a lipopeptide fragment from influenza virus a hemagglutinin.

Hemagglutinin from influenza virus A is a S-palmitoylated lipoglycoprotein in which the lipid groups are thought to influence the interaction between cell membrane and capsid during budding of viral offspring as well as fusion processes of the viral membrane with the endosome after entry of the viral particle into the cell. The paper describes the development of a method for the synthesis of characteristic lipidated hemagglutinin derived peptides which additionally carry the fluorescent 7-nitrobenz-2oxa-1,3-diazole (NBD) group. To achieve this goal the enzyme-sensitive para-phenylacetoxybenzyloxycarbonyl (PAOB) ester was developed. It is cleaved from the peptides and lipidated peptides under very mild conditions and with complete selectivity by treatment with the enzyme penicillin G acylase; this results in the formation of a phenolate. This intermediate spontaneously undergoes fragmentation thereby releasing the desired carboxylates. The combined use of this enzyme-labile fragmenting ester with the acid-labile Boc group, the Pd(0)-sensitive allyl ester and the corresponding Aloc urethane gave access to a mono-S-palmitoylated and a doubly S-palmitoylated NBD-labelled hemagglutinin peptide. The binding of these lipopeptides to model membranes was analyzed in a biophysical setup monitoring the transfer of fluorescent-labelled lipopeptide from vesicles containing the non-exchangeable fluorescence quencher Rho-DHPE to quencher-free vesicles. The experiments demonstrate that one lipid group is not sufficient for quasi-irreversible membrane insertion of lipidated peptides. This is, however, achieved by introduction of the bis-palmitoyl anchor. The intervesicle transfer always implies release of peptides localized at the outer face of the vesicles into solution followed by diffusion to and insertion into acceptor vesicles. For peptides bound at the inner face of the vesicle membrane, however, an additional flip-flop diffusion to the outer face has to occur beforehand. The kinetics of these processes were estimated by fast chemical quench of the outside fluorophores by sodium dithionite.

Amino Acid Sequence↗
PubMed2001

Synthesis and biological investigations of 5-substituted pyrimidine nucleosides coupled to a dihydropyridine/pyridinium salt redox chemical delivery system.

The syntheses, antiviral activities, and partition coefficients (P) of 3'-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-coupled nucleosides are described. These novel compounds were designed in an effort to enhance the lipophilicity, and thereby the delivery to the CNS, without compromising the anti-HSV-1 activity of the parental nucleosides. We have previously reported the synthesis of 3'-O-(1-methyl-1,4-dihydropyridyl-3- carbonyl) analogs of 5-iodo-(5), 5-vinyl-(6), and (E)-5-(2-iodovinyl)-2'-deoxyuridines (7). We now report the synthesis of 5-iodo-3'-O-(1-methyl-1,4-dihydropyridyl-3- carbonyl)-5'-O-acetyl-2'-deoxyuridine (15) and 3'-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-2'-deoxyuridine (17). Quarternization of the 3'-O-(3-pyridylcarbonyl) compounds (10,12) using iodomethane afforded the corresponding 1-methyl pyridinium salts (13,14) which were reduced with sodium dithionite to yield the corresponding 3'-O-1-methyl-1,4-dihydropyridyl-3-carbonyl compounds (15,16). The deprotection of 3'-O-(1-methyl-1,4-dihydropyridyl- 3-carbonyl)-5'-O-t-butyldimethylsilyl-2'-deoxyuridine (16) with Bu4N+F- afforded 3'-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-2'-deoxyuridine (17). Compounds 5-7 and 15 were evaluated for their antiviral activity in vitro against HSV-1, HSV-2, HCMV, and VZV, and were found to retain anti-HSV-1, HSV-2 and VZV activity as compared to their parental nucleosides (1-3). In addition, the cellular toxicity of 3'-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-coupled compounds (5-7 and 15) was found to be lower than the parent nucleosides. The lipophilicity of compounds (5-7,15,17) are enhanced substantially, compared to the parent nucleosides, as indicated by an increase in corresponding P values (1-octanol-water) upon replacement of the C-3' hydroxyl by 1-methyl-1,4-dihydropyridyl-3-carbonyl moiety.

Antiviral Agents↗
PubMed1991

Ultrastructural localization of tartrate-resistant acid phosphatase (purple acid phosphatase) activity in chicken cartilage and bone.

Tartrate-resistant acid adenosine triphosphatase activity at pH 6.5, using a lead-salt method, was localized at light and electron microscopic levels in cartilage and bone matrices, osteoclasts, and chondroclasts. Cartilage matrix staining occurred after vascular invasion of the growth plate. In osteoclasts, activity was present in lysosomes, extracellular ruffled border channels, and the underlying cartilage and bone matrices. Staining artifacts occurred at lower pH levels (pH 5.4, 5.0). Adenosine diphosphate, p-nitrophenylphosphate, thiamine pyrophosphate, and alpha-naphthylphosphate also acted as substrates; but no activity was observed when adenosine monophosphate, adenylate-(beta, gamma-methylene) diphosphate, and beta-glycerophosphate were used. The activity was inhibited by NaF, dithionite, and a high concentration of p-chloromercuribenzoic acid, and activated by simultaneous addition of FeCl2 and ascorbic acid, as has been shown in biochemical studies. These histochemical results support the view that the adenosine triphosphate hydrolyzing activity at pH 6.5 is due to tartrate-resistant acid phosphatase (TRAP). There were some differences in ultrastructural localization between TRAP and tartrate-sensitive acid phosphatase (TSAP) activities in osteoclasts: TSAP activity was more intense in lysosomes and Golgi complexes and TRAP was stronger in the cartilage and bone matrices. It is suggested, therefore, that most of TRAP is in an inactive form in cells and is activated when secreted.

Acid Phosphatase↗