Studies on human uterine cervix and rat uterus using S-, X- and Q-band electron-spin-resonance spectroscopy.

In previous studies we have reported on the detection of a strong e.s.r. signal in samples of normal human cervix; the signal is much reduced or absent in samples of invasive cancer of the cervix. In order to identify the species responsible for the strong signal, we have used X-, S- and Q-band e.s.r. spectroscopy. The major signal that is detectable in ground-up samples of cervix preserved at -196 degrees C has features consistent with the presence of a peroxy free radical. Good agreement with the experimental findings was obtained by computer simulation, using values for the g-tensor of gx = 2.002, gy = 2.005 and gz = 2.036. The peroxy radical is produced on grinding the normal cervix samples to a powder under liquid N2, and appears to be formed by modification of a pre-existing oxygen-containing complex. Control experiments eliminated the possibility that the strong signals seen in frozen powders prepared from normal cervix were artefacts only of the grinding procedure. Experiments with rats in vivo and with cervix samples in vitro are consistent with the conclusion that the peroxy radical is formed by disturbing the cyclo-oxygenase system that is involved in prostaglandin synthesis.     

Studies on the photolytic breakdown of hydroperoxides and peroxidized fatty acids by using electron spin resonance spectroscopy. Spin trapping of alkoxyl and peroxyl radicals in organic solvents.

Spin trapping using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has been used to detect and distinguish between the carbon-centred, alkoxyl, and peroxyl radicals produced during the photolytic decomposition of hydroperoxides. Photolysis of tert-butyl and cumene hydroperoxides, and peroxidized fatty acids, in toluene, with low levels of u.v. light, is shown to lead to the initial production of alkoxyl radicals by homolysis of the oxygen-oxygen bond. Subsequent reaction of these radicals with excess hydroperoxide leads, by hydrogen abstraction, to the production of peroxyl radicals that can be detected as their corresponding adducts with the spin trap. Subsequent breakdown of these adducts produces alkoxyl radicals and a further species that is believed to be the oxidized spin-trap radical 5,5-dimethyl-1-pyrrolidone-2-oxyl. No evidence was obtained at low hydroperoxide concentrations, with either the cumene or lipid alkoxyl radicals, for the occurrence of beta-scission reactions; the production of low levels of carbon-centred radicals is believed to be due to the alternative reactions of hydrogen abstraction, ring closure, and/or 1,2 hydrogen shifts. Analogous experiments with 3,3,5,5-tetramethyl-1-pyrroline N-oxide (TMPO) led only to the trapping of alkoxyl radicals with no evidence for peroxyl radical adducts, this is presumably due to a decreased rate of radical addition because of increased steric hindrance.     

Conformational changes associated with transient activation of phosphorylase in glycogen particles. Studies using activity, electron-spin-resonance and phosphorus-nuclear-magnetic-resonance measurements.

1. Calcium-dependent transient phosphorylation of phorphorylase b has been monitored in a rabbit muscle glycogen particle fraction. Using a phosphorus nuclear magnetic resonance assay, the changes in concentrations of small phosphate-containing metabolites associated with this event have been measured. In addition, the conformation of phosphorylase has been monitored during transient activation by observing changes in the electron spin resonance signal from added spin-labelled phosphorylase. 2. The transient activation was associated with a loss of glucose-6-phosphate from phosphorylase b; newly formed phosphorylase a binds the nucleotides ADP, AMP, or IMP. Because of the fast interconversion of these nucleotides the species bound to phosphorylase a change throughout the process. 3. Lowering the [Mg2+] : [Ca2+] ratio during transient activation causes accumulation of ADP. Electron spin resonance data from spin-labelled phosphorylase shows that, under these conditions, ADP binding to phosphorylase a is potentiated. 4. Calcium-dependent activation in the glycogen particle fraction is compared to the activation of phosphorylase in vivo.     

An investigation of the iron-sulphur proteins of benzene dioxygenase from Pseudomonas putida by electron-spin-resonance spectroscopy.

Benzene dioxygenase from Pseudomonas putida comprises three components, namely a flavoprotein (NADH:ferredoxin oxidoreductase; Mr 81000), an intermediate electron-transfer protein, or ferredoxin (Mr 12000) with a [2Fe-2S] cluster, and a terminal dioxygenase containing two [2Fe-2S] iron-sulphur clusters (Mr 215000), which requires two additional Fe2+ atoms/molecule for oxygenase activity. The ferredoxin and the dioxygenase give e.s.r. signals in the reduced state with rhombic symmetry and average g values of 1.92 and 1.896 respectively. The mid-point redox potentials were determined by e.s.r. titration at pH 7.0 to be -155 mV and -112 mV respectively. The signal from the dioxygenase shows pronounced g anisotropy and most closely resembles those of 4-methoxybenzoate mono-oxygenase from Pseudomonas putida and the [2Fe-2S] 'Rieske' proteins of the quinone-cytochrome c region of electron-transport chains of respiration and photosynthesis.     

Cu2+ probe of metal-ion binding sites in melanin using electron paramagentic resonance spectroscopy. II. Natural melanin

The isotope ⁶³Cu²⁺ has been used to probe the metal-ion binding sites of natural melanin from the choroid of bovine eyes using electron paramagnetic spectroscopy. Samples were in aqueous media over a wide range of pH values. At pH < 7, binding is to monodentate carboxyl complexes and to bidentate nitrogen-carboxyl complexes just as in synthetic melanin. At pH > 7 binding is to phenolic hydroxyl groups, but the number of such sites is much less than in synthetic melanin and there are indications of a superimposed spectrum of another site. At high pH, a signal unlike any found in synthetic melanin was observed with either three or four nitrogen ligands. A number of experiments indicate that natural melanin is 50% protein by weight. Metal-ion binding sites are the same with and without protein although with some differences in relative populations.     

Characterization of laser produced plasma using laser induced breakdown spectroscopy

The plasma parameter studies of the Nd:YAG (neodymium-doped yttrium aluminum garnet, Nd:Y₃Al₁₅O₁₂) crystal by using the fundamental (1064 nm) and second (532 nm) harmonics of Nd:YAG laser are reported. The electron temperature (T _e ) and electron number density (N _e) were determined using the Boltzmann plot method and the Stark-broadened line profile, respectively. An increase in the plasma parameters have been observed with an increase in the laser irradiance for both laser modes. The electron temperatures were calculated in the range of 0.53–0.66 eV for 1064 nm and 0.47–0.60 eV for 532 nm, and the electron number densities were determined in the range of 7.43 × 10¹⁵–3.27 × 10¹⁶ cm^?3 for 1064 nm and 1.35 × 10¹⁶–3.97 × 10¹⁶ cm^?3 for 532 nm in the studied irradiance range of 1.19–12.5 GW/cm². However, the spatial evolution of the plasma parameters investigated up to 2.75 mm away from the target surface at a fixed laser irradiance of 6.51 GW/cm2 showed a decreasing trend. In addition, the estimated values of the inverse bremsstrahlung (IB) absorption coefficients at both laser wavelengths showed that the IB process is dominant for the 1064-nm laser.     

Cu2+ probe of metal-ion binding sites in melanin using electron paramagnetic resonance spectroscopy. I. Synthetic melanins

The isotope ⁶³Cu²⁺ has been used to probe the metal-ion binding sites of synthetic (autoxidized) catechol and 3,4-dihydroxyphenylalanine melanins using electron paramagnetic resonance spectroscopy. Samples were in aqueous media over a wide range of pH values. Assignments of the structures of the melanin-copper complexes are based in part on model studies of the complexes formed with melanin precursors, catechol and 3,4-dihydroxyphenylalanine, and with phenanthroline. Nearly all complexes involve just one or two ligands from melanin. In catechol melanin below pH 5.0, complexes with carboxyl groups are formed; above 6.0, Cu²⁺ forms complexes with phenolic hydroxyl groups. These same complexes were found in 3,4-dihydroxyphenylalanine melanin and binding of Cu²⁺ at amino acid type sites also was detected. After partial reduction of copper ions bound to 3,4-dihydroxyphenylalanine melanin, a weak signal of copper with four melanin ligands (oxygen and nitrogen in various combinations) was observed.     

Study of the respiratory chain in Micrococcus luteus (lysodeikticus) by electron-spin-resonance spectroscopy

Low-temperature electron spin resonance spectroscopy was used to investigate the redox centres of Micrococcus luteus membranes. Three different types of iron-sulphur centres were distinguished. Two of these, a [4Fe-4S]3+-type cluster giving rise to a signal at g = 2.01 in the oxidized state and a [2Fe-2S] cluster with a spectrum at g = 2.03 and 1.93 in the reduced state, were attributable to succinate dehydrogenase. Another, generating signals in the reduced state at g = 2.027, 1.90 and 1.78 was identified as a 'Rieske' iron-sulphur centre. This latter cluster had a mid-point potential (pH 7.0) of +130 mV. In addition, signals characteristic of high-spin ferric haem (g = 6.20), low-spin ferric haem (g = 3.67, 3.36 and 3.01) and Cu2+ (g = 2.18 and 2.02) were also detected. The ferric-haem features, together with the Cu2+ and 'Rieske' centres, were enriched in membrane residues insoluble in Triton X-100, which are known from difference spectroscopy to contain cytochromes b-560, c-550 and a-601 (aa3 oxidase). The signals demonstrated by electron spin resonance for M. luteus membranes showed marked similarities to those documented for the complexes II, III, and IV of mitochondria. However, signals analogous to complex I (NADH-ubiquinone reductase) could not be demonstrated for M. luteus membranes.     

Studies on the structure of actin gels using time correlation spectroscopy of fluorescent beads.

Fluorescence correlation spectroscopy (FCS) has been used to measure the diffusion of fluorescently labeled beads in solutions of polymerized actin or buffer. The results, obtained at actin concentrations of 1 mg/ml, show that small beads (0.09 micron in diameter) diffuse nearly as rapidly in the actin gel as in buffer, whereas the largest beads tested (0.5 micron in diameter) are immobilized. Measured autocorrelation times for motions of beads with intermediate sizes show that the diffusion is retarded (relative to buffer) and that the time behavior cannot be represented as a single diffusive process. In addition to the retarded diffusion observed over distances > 1 micron, 0.23-micron beads also show a faster motion over smaller distances. Based on the measured rate of this faster motion, we estimate that the beads may be constrained within a cage approximately 0.67 micron on a side, equal to a filament length of approximately 250 subunits. Fluorescence correlation spectroscopy measurements made in the same small spot (radius of 1.4 microns) of the gel vary over time. From the variations of both the autocorrelation functions and the mean fluorescence, we conclude that, corresponding to a spatial scale of 1.4 microns, the actin gel is a dynamic structure with slow rearrangement of the gel occurring over periods of 20-50 s at 21-22 degrees C. This rearrangement may result from local reorganization of the actin matrix. Data for the retardation of beads by the actin gel are consistent with a detailed theory of the diffusion of particles through solutions of rigid rods that have longitudinal diffusion coefficients much less than that of the particles (Ogston, A. G., B. N. Preston, and J. D. Wells. 1973. Proc. R. Soc. Lond. A. 333:297-316).     

Chemiluminescence during lipoxygenase-catalysed oxygenation of linoleic acid.

Contrary to earlier observations (7) the present investigation shows that light emission from the lipoxygenase-catalysed oxygenation of linoleic acid can be readily measured in the absence of luminol with standard liquid scintillation counting equipment. The quenching effect of superoxide dismutase suggests superoxide $\text{(O}{}_2{}^{\mathrm{<mtext>?</mtext>}}\text{)}$ to play a key role in this process.     

Studies on the role of vitamin E in the oxidation of blood components by fatty hydroperoxides.

Studies are reported on the oxidation of vitamin E and changes in lipid and fatty acid composition of rat blood components incubated in vitro with hydroperoxides prepared from autoxidized methyl linoleate. Red blood cells, plasma, serum, and hemoglobin free stroma were incubated at 37 °C with suspensions of linoleate hydroperoxide in Tris buffer at pH 7.4. The RBC were destroyed and substances with excitation-fluorescent properties were produced. Phosphatidylethanolamine, vitamin E and unsaturated fatty acids were oxidized in the reaction. Among the reaction products were substances that gave a positive thiobarbituric acid value, tocoquinone, and an unidentified substance isolated in the nonsaponifiable fraction of the lipid extract of the hemolyzed red cells. The reaction of linoleate hydroperoxide with stroma was similar to that with red blood cells and the same products were observed. In contrast there was little reaction of linoleate hydroperoxide with vitamin E or lipids of the serum or plasma in the absence of red blood cells. The destruction of the red blood cells appeared to be closely related to the oxidation of vitamin E indicating that the strong antioxygenic action of vitamin E in vivo was due to its particular form or structural orientation in the red cell membrane.     

Studies on the primary structure of short polysaccharides using SEC MALDI mass spectroscopy

The introduction of size-exclusion chromatography (SEC) analysis of polysaccharides prior to MALDI mass spectroscopy accounts for the determination of the molecular mass of the repeating unit when neutral homopolymers are investigated. In the case of natural polysaccharides characterised by more complicated structural features (presence of non-carbohydrate substituents, charged groups, etc.), this mass value usually is in agreement with more than one sugar composition. Therefore, it is not sufficient to give the correct monosaccharidic composition of the polysaccharide investigated. To solve this problem, MALDI spectra were recorded on the permethylated sample and post-source decay experiments were performed on precursor ions. In this way, the composition (in terms of Hex, HexNAc, etc.), size and sequence of the repeating unit were determined.     

Studies on the biosynthesis of 16-membered macrolide antibiotics using carbon-13 nuclear magnetic resonance spectroscopy.

The origin of the skeletal carbons in the lactone ring of 16-membered macrolide antiobiotics has been studied. 13C-labeled antibiotics leucomycin and tylosin, have been obtained from the culture broth of Streptomyces kitasatoensis 66-14-3 and Streptomyces fradiae C-373, respectively in the presence of appropriate 13C-labeled precursors, and 13C NMR spectra of the antibiotics thus obtained have been measured. It was shown that the aglycone of leucomycin A3 is derived from five acetates, one propionate, one butyrate, and an unknown precursor corresponding to two carbons. The formyl carbon which is characteristic of the basic 16-membered macrolides orginates from C-4 butyrate. On the other hand, the aglycone of tylosin is formed from two acetates, five propionates and one butyrate. Butyric acid and ethylmalonic acid are metabolized to propionyl-CoA or methylmolonyl-CoA through a pathway involving methylmalonyl-CoA mutase, and subsequently incorporated into the lactone ring of tylosin.     

Cyclic peroxides from a soya lipoxygenase-catalysed oxygenation of methyl linolenate.

Incubation of methyl linolenate with an aqueous extract of soyabean flour at neutral pH gives the hydroperoxyendoperoxides 16-hydroxyperoxy-13,15-endoperoxylinolenate and 9-hydroxyperoxy-10,12-endoperoxylinolenate as the principal oxygenation products in addition to monohydroperoxides. Lipoxygenase I (EC 1.13.11.12) does not catalyse the oxygenation under this condition. The enzyme contributing most to the formation of the hydroperoxyendoperoxides is assumed to be a high molecular mass lipoxygenase aggregate.     

Studies of the retrogradation process for various starch gels using Raman spectroscopy

The retrogradation of untreated wild-type starches (potato, maize, and wheat), waxy maize starches, and one pregelatinized, modified amylose-rich starch was investigated continuously using Raman spectroscopy. The method detects conformational changes due to the multi-stage retrogradation, the rate of which differs between the starches. The pregelatinized, modified amylose-rich starch shows all stages of retrogradation in the course of its Raman spectra. In comparison to amylose, the retrogradation of amylopectin is faster at the beginning of the measurements and slower in the later stages. The untreated starches can be ranked in the order of their rate of retrogradation as follows: potato>maize>wheat.