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Electron transfer to 5-bromouracil (5-BrU) from nucleobase (N) electron adducts (and their protonated forms) has been studied by product analysis and pulse radiolysis. When an electron is transferred to 5-BrU, the ensuing 5-BrU radical anion rapidly loses a bromide ion; the uracilyl radical thus formed reacts with added t-butanol, yielding uracil. From the uracil yields measured as the function of [N]/[5-BrU] after gamma-radiolysis of Ar-saturated solutions it is concluded that thymine and adenine electron adducts and their heteroatomprotonated forms transfer electrons quantitatively to 5-BrU. Like the electron adduct of adenine, those of cytosine and guanine are rapidly protonated by water. The (protonated) electron adduct of guanine does not transfer an electron to 5-BrU, and in the case of the (protonated) cytosine electron adduct only partial electron transfer is observed. The results can be modelled if the protonated electron adduct (protonated at N(3) or at the amino group) of cytosine, CH., which can transfer its electron to 5-BrU (k approximately 2 x 10(7) dm3 mol-1 s-1) is transformed in a slow tautomerization reaction (k approximately 2.5 x +/- 10(3) s-1) into another form C'H. (possibly protonated at C(6) or C(5)) which does not transfer an electron to 5-BrU. There is also electron transfer from the electron adduct of thymine to cytosine and guanine which serve as electron sinks. The rate constant of electron transfer from the thymine electron adduct to cytosine is about 250 times greater than that of the reverse reaction. The heteroatom-protonated electron-adduct of thymidine transfers an electron to 5-BrU more slowly (k = 2.3 x 10(7) dm3 mol-1 s-1) than the electron-adduct itself (k = 7.2 x 10(8) dm3 mol-1 s-1). Phosphate buffer-induced protonation of the electron-adduct of thymine at carbon (C(6)) prevents electron transfer to 5-BrU. Such phosphate catalysis is also observed as an intramolecular process (k approximately 2 x 10(4) s-1) with thymidine-5'-phosphate but not with the 3'-phosphate. Phosphate-induced protonation at carbon also reduces transfer efficiency for the electron adducts of dinucleoside phosphates such as dTpdT and dTpdA. The data raise the question whether in DNA the guanine moiety may act as the ultimate sink of the electron in competition with other processes such as protonation at C(6) of the thymine electron adduct.
An e.p.r. spectrum of the reduced form of the electron-transport component (X), thought to be the primary electron acceptor of Photosystem I, was obtained. By using line-shape simulations of this component and the free-radical e.p.r. signal I of the oxidized reaction-centre chlorophyll (P700), it was possible to determine the ratio of the number of electron spins to which these signals correspond in Photosystem-I particles under a variety of conditions. On illumination at cryogenic temperatures of Photosystem-I preparations, in which both bound iron-sulphur centres A and B were reduced, the measured ratio of free radical to component X varied between 1.04 and 2.23, with an average value of 1.54 +/- 0.18 where a Gaussian line-shape is assumed for the component-X signal in the simulation. The error in this measurement is estimated to be up to 50%. In a similar way component X and centre A of the bound iron-sulphur protein were quantified, the ratio between these two components varying between 1.26 and 0.61 with an average value of 0.75 +/- 0.06. These results indicate that the quantitative relationship, in terms of net electron spins, between centre A, component X and P700 is of the order to be expected if component X is indeed the primary electron acceptor in Photosystem I and a component of the photosynthetic electron-transport chain.
A mathematical analysis is described which measures the effects of actinic light intensity and concentration of an artificial electron donor on the steady-state light-induced redox level of a reaction-center pigment (e.g. P-700) and on the overall light-induced electron flux (e.g. reduction of NADP+). The analysis led to a formulation (somewhat similar to the Michaelis-Menten equation for enzyme kinetics) in which a parameter, I1/2, is defined as the actinic light intensity that, at a given concentration of electron donro, renders the reaction-center pigment half oxidized and half reduced. To determine the role of a presumed reaction-center pigment, I1/2 is compared with another parameter, equivalent to I1/2, that is obtained independently of the reaciton-center pigment by measuring the effect of actinic light intensity and concentration of electron donor on the overall electron flow. The theory was tested and validated in a model system with spinach Photosystem I chloroplast fragments by measurements of photooxidation of P-700 and light-induced reduction of NADP+ by reduced 2,6-dichlorophenolindophenol. A possible extension of this mathematical analysis to more general electron-transport systems is discussed.
In order to describe initial events in enamel mineralization and to help characterize inorganic-organic interactions in this tissue, the earliest rod and interrod enamel in mandibular incisors from normal young adult (100 gm) rats, perfused with 100% ethylene glycol, has been studied by transmission electron microscopy, selected area electron diffraction, and high-spatial-resolution electron probe microanalysis. Diffraction and probe data were correlated precisely from the same extracellular regions of the tissue. Sites were examined progressively as a function of location a) from the most recently deposited enamel adjacent to ameloblasts toward the dentin-enamel junction and b) from the apical portion of the tooth longitudinally toward its incisal end. Electron diffraction patterns consistent with that of a poorly crystalline hydroxyapatite were generated at all locations. Diffraction characteristics changed only slightly toward that of more crystalline hydroxyapatite at different locations. Earliest apical enamel generated molar Ca/P ratios in a range of 0.99-1.46 (average 1.24 +/- 0.15). Molar Ca/P ratios of the first enamel interrod elements increased from approximately 1.24 at ameloblast-enamel boundaries to approximately 1.40 at the dentin-enamel junction, small changes corresponding to those observed in electron diffraction characteristics.
Oxidation-reduction potentiometry was carried out on Rhodopseudomonas viridis chromatophores. Measurements of e.p.r. signals of the semiquinone-iron type at g=1.82 have revealed a more complex situation than previously reported. The presence of three different components is indicated. The midpoint potential (E(m)) of the primary acceptor quinone/semiquinone couple was found to be approx. -165mV at pH10, with a pK being reached at around pH7.5. The primary acceptor also accepts a second electron with an E(m) of -525mV, but this redox transition exhibits a hysteresis effect. Interaction effects indicate the presence of another component with E(m) values at pH10 of approx. -165mV (pK reached at around pH7.5) for single reduction and -350mV (pK at pH10 or greater) for double reduction. It is suggested that this component is the secondary acceptor. Another semiquinone-iron-type component which gives a g=1.82 signal is also present. This component is distinguishable from the primary acceptor by its e.p.r. spectrum, which shows a double peak at g=1.82 and a g(x) line at g=1.76. This component has E(m) values at pH10 for single and double reduction of -15mV and approx. -150mV respectively. Both of these E(m) values are pH-dependent. The presence of an interaction between this component and the photoreduced primary acceptor indicates the close proximity of these components. However, the midpoint potential of this component indicates a function as a secondary electron-transport component rather than an electron acceptor in the reaction centre. The dependence of the bacteriopheophytin intermediate (I) doublet e.p.r. signal on the presence of the semiquinone-iron form of the primary acceptor is demonstrated. The midpoint potential of the I/I(-) couple is estimated to be lower than -600mV.
The dose due to Bremsstrahlung in stationary electron beams of nominal energies in the range 6-20 MeV is typically between 1-7% of the maximum dose and is usually not clinically significant. However, in treatments using rotational or multiple electron beams where the x-ray dose from several beams is added the x-ray dose will reach much higher proportions and will be of clinical significance. Moreover, this dose often is located in normal tissue beyond the target volume. Reduction of this x-ray dose is therefore desirable. In the present study a reduction of the x-ray component of electron beams produced by a Clinac 2100C accelerator by a change of the transmission ion chamber and scattering foils is reported. A reduction in Bremsstrahlung of up to 50% can be achieved.
The mode of correction of the isodose curves from telecentric electron pendulous technique using a constant patient radius rp = 30 cm (Isodosenatlas, Siemens, 1973) is represented with regard to its application in patients with diverging surface contours. Correction is possible by two different methods: 1st by experimental determination of an air gap factor for the shift of isodoses, and 2nd by two factors depending on the focus-skin distance and on the angle of incidence of the electron beam. Determination of the factors is performed either by means of fixed fields measured by vertical and at oblique incidence of the beam and a depth dose distribution measured at the central axis, with oblique incidence of the electrons.
The metal secretory granules of Langerhans' islets of white rats were examined after histochemical detection by sulfide silver staining method as well as by conventional electron microscopy and high voltage electron microscopy. Zinc and calcium were determined with electron probe x-ray-microanalysis. The metals are located at the inside of cytoplasmatic vacuoles, a result, we discuss in connection with the release of pancreatic hormones.
The results of a study of 90 patients are presented. Intrapulmonary mineral deposits were characterized by electron diffraction and electron probe microanalysis. Using this method, pneumoconioses may be distinguidhed from other pneumopathies. In cases of pneumoconiosis, there exists a specific relationship between the etiology of the dust exposure and the crystallographic characteristics of the intrapulmonary deposits. The nature of the deposits may be indicative of a specific type of pneumoconiosis. This method is particularly useful in differentiating between asbestos bodies and ferruginous bodies. The value of the method in general and its importance in the study of pneumoconiosis are discussed.
Electron spin lattice relaxation times (T1) and the phase memory times (Tm) were obtained for the synthetic melanin system from 3-hydroxytyrosine (dopa) by means of electron spin echo spectroscopy at 77 degrees K. Saturation behavior of the ESR spectra of melanins in melanin-containing tissue and of the synthetic melanin was also determined at the same temperature. The spin lattice relaxation time and the spectral diffusion time of the synthetic melanin are very long (4.3 ms and 101 microseconds, respectively, in the solid state), and the ESR signal saturates readily at low microwave powers. On the other hand, ESR spectra of natural melanins from the tissues chosen for this study, as well as those of synthetic melanins which contain Fe3+ of g = 4.3 and Mn2+ of g = 2, are relatively difficult to saturate compared with samples without such metal ions. These results show clearly that a large part of those two metal ions in sites responsible for the ESR spectral components with these particular g values are coordinated to melanin in melanin-containing tissue, and modify the magnetic relaxation behavior of the melanin. Accumulations of these metal ions in melanins are different from system to system, and they increase in the order: hair (black), retina and choroid (brown), malignant melanoma of eye and skin, and lentigo and nevus of skin.
One consequence of radiation damage to DNA is the conversion of thymine to 5-hydroxymethyluracil (HMU). In order to sensitively detect this DNA adduct by gas chromatography (GC) or high-performance liquid chromatography (HPLC) with electron-capture detection techniques, it is necessary to derivatize it. This study was designed to select an optimum ester derivative of the aliphatic hydroxyl group on HMU. N1, N3-Bis(pentafluorobenzyl)-HMU was formed as a parent derivative, and from this a series of esters. Also O-pentafluorobenzyl and O-tetrafluorobenzyl ether derivatives were prepared. Of the esters the pivalyl derivative was the best choice because it formed easily, was relatively stable to aqueous hydrolysis (t 1/2 = 9.8 days at pH 11.5, 24 degrees C) and gave a response at fmol levels by GC and LC comparable to that of the ethers. Unanticipated was a good response as well for the parent derivative, a free hydroxyl compound, by GC and LC at this level. The work also demonstrates a high performance by LC-electron-capture negative ion mass spectrometry with a belt interface for the trace detection of derivatives of this type.
Several methods of tissue preparation and different modes of operation of the scanning electron microscope were used to study the ultrastructure of rat liver. Rat livers were perfusion fixed with buffered 2 per cent paraformaldehyde or a mixture of 1.5 per cent paraformaldehyde and 1 per cent glutaraldehyde and processed as follows. Tissue blocks were postfixed in buffered 2 per cent osmium tetroxide followed sequentially by the ligand-mediated osmium binding technique, dehydration and cryofracture in ethanol, and critical point drying. They were then examined without metal coating in the scanning electron microscope operating in the secondary electron and backscattered electron modes. Fifty-micrometer sections were cut with a tissue sectioner, stained with lead citrate, postfixed with osmium, dehydrated, critical point dried, and examined in the secondary electron and back-scattered electron modes. Frozen sections (0.25 to 0.75 mum. thick) were cut by the method of Tokuyasu (Toluyasu KT: J Cell Biol 57:551, 1973) and their scanning transmission electron microscope images were examined either with a scanning transmission electron microscope detector or with a conversion stub using the secondary electron detector. Secondary electron images of the liver prepared by ligand-mediated osmium binding and subsequent cryofracture revealed such intracellular structures as cisternae of the endoplasmic reticulum, lysosomes, mitochondria, lipid droplets, nucleolus and nuclear chromatin, as well as the usual surface morphology, Lipocytes in the perisinusoidal space were readily identified. Backscattered electron images. Unembedded frozen sections had little drying artifact and were virtually free of freezing damage. The scanning transmission electron microscope image revealed those organelles visualized by the secondary electron mode in the ligand-mediated osmium binding-treated tissue.
The reduction by L-lactate of the prosthetic groups of flavocytochrome b2 (L-lactate cytochrome c oxidoreductase from aerobic yeast, a tetrameric molecule containing one haem and one flavin mononucleotide per protomer) was reinvestigated. It was confirmed that the enzyme ultimately takes up 3 electrons per protomer from this 2-electron donor. Stopped-flow absorbance data at an haem isosbestic point to follow the oxidized flavin and in a haem band indicate that, under the conditions used, haem and flavin reduction time courses are indistinguishable, both being biphasic (phases I and II). Comparison with electron paramagnetic resonance data (Fe3+ haem and flavosemiquinone signals) led to a complete description at 24 degrees C of the time courses of the various reduction states of the prosthetic groups. It has been previously demonstrated (Morton and Sturtevant, 1964) that, after the formation of the enzyme-substrate complex, the electron transfer to the enzyme takes place as the first and rate-limiting step of the turnover. In the present study, an initial burst of fully reduced flavin, of small amplitude, is detected at the very beginning of phase I (before 6 ms). The redox forms which accumulate thereafter till the end of phase I (30-35 ms) are the reduced haem (up to 80%), the flavin semiquinone (up to 50%) and the fully reduced flavin (from 25% up to 35%); the total of electrons distributed at the end of phase I is about 2 per protomer meaning that, in this phase, each enzyme site acts as a 2-electron and not a 3-electron acceptor. A 2-electron flow as the limiting step during phase I with the rate constant kI accounts for the steady-state electron flow during catalysis. Phase I is followed by the much slower phase II which corresponds to the entry of the third electron and cannot be involved in the turnover. The interpretation of the results are given as a scheme, with the proper rate constants, allowing a satisfactory fitting of experimental data by simulation. Among the elementary steps required are a rapid distribution of one electron from reduced flavin to the haem, a rapid interprotomers dismutation between couples of flavin semiquinone regenerating two oxidized flavin per tetramer. The very low reactivity of the latter for the entry of the third electron per protomer is tentatively explained by the occurrence of a slow additional step limiting the final reduction reaction. It was observed that, over phase I and the beginning of phase II, from 15 to 200 ms, all the redox species remain apparently under equilibrium conditions. Parallel studies (titrations of flavocytochrome b2 by L-lactate) showed that the set of equilibrium parameters relative to haem and flavin species is significantly different in the "final" equilibrium (after 30 s) from that in the time interval 15-200 ms. Such an anomaly suggests a conformation change takes place very slowly in the molecule after the acceptance of the first two electrons per protomer.
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Part of the catalytic function of xanthine oxidase (XO) involves the transfer of two electrons from a substrate to a molybdenum ion on the enzyme followed by equilibration of these electrons among other electron resting sites on the enzyme. The electrons are removed from the enzyme at a flavin by oxygen to form hydrogen peroxide. This paper considers mechanisms which allow the electrons to equilibrate between the different resting sites on the enzyme. The mechanisms are chosen to be consistent with known properties of the enzyme (relative reduction potentials, electron transfer rates, and the estimated separation of these resting sites). Tunneling appears to be a good candidate to account for most of the electron transport. It is shown that the XO electron transport system is similar in many respects to sections of mitochondrial electron transport chains and can serve as a nice model for parts of these more complicated biological electron transport systems.
The oxidation-reduction potential, E2, for the couple oxidized lipoamide dehydrogenase/2-electron reduced lipoamide dehydrogenase has been determined by measurement of equilibria of these enzyme species with lipoamide and dihydrolipoamide or with oxidized and reduced azine dyes. E2 is -0.280 V at pH 7, and deltaE2/deltapH is -0.06 V in the pH range 5.5 to 7.6. Values for E1, the oxidation-reduction potential for the couple 2-electron reduced enzyme/4-electron reduced enzyme, were obtained from measurements of the extent of dismutation of 2-electron reduced enzyme to form mixtures containing oxidized and 4-electron reduced enzyme. E1 is -0.346 V at pH 7, and deltaE1/deltapH is -0.06 V in the pH range 5.7 to 7.6. Spectra of oxidized enzyme and 4-electron reduced enzyme do not show variations with pH over this range, but the spectrum of the 2-electron reduced enzyme is pH-dependent, with the molar extinction at 530 nm changing from 3250 M-1 cm-1 at pH 8 to 2050 M-1 cm-1 at pH 5.2. The pH-dependent changes which are observed in the absorption properties of the 2-electron reduced enzyme are consistent with the disappearance of a charge transfer complex between an amino acid side chain and the oxidized flavin at the lower pH values, with the apparent pK of the side chain at pH 5. It has been suggested that the 530 nm absorbance of 2-electron reduced enzyme is due to a charge transfer complex between thiolate anion and oxidized flavin, and we propose that the thiolate anion is stabilized by interaction with a protonated base. The thermodynamic data predict that the amount of 4-electron reduced enzyme formed when the enzyme is reduced by excess NADH will be pH-dependent, with the greatest amounts seen at low pH values. These data support earlier evidence (Matthews, R.G., Wilkinson, K.D., Ballou, D,P., and Williams, C.H., Jr. (1976) in Flavins and Flavoproteins (Singer, T.P., ed) pp. 464-472; Elsevier Scientific Publishing Co., Amsterdam) that the role of NAD+ in the NADH-lipoamide reductase reaction catalyzed by lipoamide dehydrogenase is to prevent accumulation of inactive 4-electron reduced enzyme by simple reversal of the reduction of 2-electron reduced enzyme by NADH.
Results are presented which demonstrate that the 2-electron-reduced lipoamide dehydrogenase (EC 1.6.4.3) from Escherichia coli is a mixture of species. In catalysis, this enzyme cycles between the oxidized and the 2-electron-reduced forms. Three spectrally distinct species are indicated in the pH range 5.8 to 8.0 from measurements of the fluorescence and visible spectra during dithionite titration. These have the following properties. 1) A fluorescent form where the FAD is oxidized and the active center disulfide is reduced. This species is unable to charge transfer and predominates at low pH. 2) A form in which there is a facile charge transfer between thiolate and FAD (epsilon530 - 3300 M-1 cm-1). This species, which predominates at high pH, is very similar to the 2-electron-reduced pig heart enzyme at high pH. 3) A form where the flavin is reduced and the disulfide is oxidized. The spectra of these three species have been determined. Anaerobic reduction of the enzyme with stoichiometric dihydrolipoamide leads to the formation of the charge transfer species in less than 1 s. Subsequently, in a process requiring about 12 s, the charge transfer complex relaxes to a mixture of species observed in dithionite titrations. The pH dependence of the oxidation-reduction potential, the fluorescence, the charge transfer absorbance (530 nm), and the 455 nm absorbance indicates the presence of a base which is able to stabilize the thiolate anion generated upon reduction of the active center disulfide. The pH dependence of the oxidation-reduction potential indicates that the reduction of the enzyme by dihydrolipoamide involves 2 protons as well as 2 electrons. These potentials are somewhat more positive than those determined for the pig heart enzyme and thus explain the ready further reduction of the E. coli enzyme to the 4-electron-reduced enzyme. The pH-independent formation constant (Kf) for the disproportionation of 2-electron-reduced enzyme (2EH2 in equilibrium E + EH4) is about 55 as calculated from dithionite titrations. Therefore at equilibrium there is about 80% 2-electron-reduced enzyme, 1-% oxidized enzyme, and 10% 4-electron-reduced enzyme. The spectrum of fully formed 2-electron-reduced enzyme has been calculated at several pH values from these data. The results confirm the previous conclusion that lipoamide dehydrogenase from E. coli is qualitatively similar to the pig heart enzyme, differing only in certain quantitative features such as the distribution between the various forms at the 2-electron-reduced level.