Martyn C. R. Symons

Electron spin resonance studies of intact mammalian skeletal muscle

Samples of skeletal muscle from mice, rats and man have been examined by conventional electron spin resonance techniques. One major free-radical signal with g value 2.0036-2.004 was detected in all intact muscle samples and homogenates at 77 K whereas this signal was not seen at room temperature. Other less prominant signals were also detected. Thirty minutes of excessive contractile activity of rat hind limb muscles was found to result in a leakage of intracellular creatine kinase enzyme into the blood plasma and also produced an average 70% increase in the amplitude of the major electron spin resonance signal. These data support the hypothesis that increased free-radical activity may play some role in muscle damage caused by extensive muscular activity.

Molecular radical models for the muonium centres in solids

Abstract Molecular radical models are presented for the various muonium-related paramagnetic centres observed in the elemental and compound semiconductors, and for those which are currently being reported in other crystalline solids, notably halides. The measured coupling constants are shown to place tight constraints on the intrinsic geometries which need be considered, i.e. on the crystallographic sites and local relaxation of the lattice, as well as on the nature of the singly occupied molecular orbital. The role of tunnelling states is emphasized.

Effects of ionizing radiation on deoxyribonucleic acid and related systems. Part 1. The rôle of oxygen

Exposure of aqueous solutions of calf-thymus DNA to 60Co γ-rays at 77 K results in the formation of equal yields of G+ and T– centres, detected by e.s.r. spectroscopy. In the presence of oxygen, O2– ions are also formed at the expense of T–, the primary yield of G+ centres being unaffected. On annealing above 77 K the e.s.r. features for O2– ions were initially modified as a result of changes in solvation, and then, at ca. 193 K, they were lost irreversibly. A large growth in signals due to RO2· radicals was observed simultaneously. These radicals are formed from T– anions or the protonated form, TH, and probably also indirectly from G+ radicals. In parallel experiments, under comparable conditions, we have analysed for single and double stand breaks using plasmid DNA (pBR 322). Both single and double strand breaks persist under conditions in which G+ and T– are the only detectable primary radical products. The presence of oxygen causes only a slight increase in damage to DNA.

Electron-loss and electron-capture processes in irradiated systems

The use of ionizing radiation as a method for electron extraction and electron attachment under conditions in which the products are matrix-isolated are outlined, and the utility and simplicity of the method is stressed. A range of electron-loss and electron-gain mechanisms are discussed in terms of molecular relaxation, bond-making and bond-breaking processes, with particular reference to solvation effects.

The hydration of monosaccharides—an NMR study

At temperatures close to 0°C proton exchange between sugar hydroxyl groups and water is slow, and separate proton resonance peaks can be detected for the hydroxyl protons. All are shifted downfield of the water resonance, the anomeric hydroxyl proton shift being the greatest. Axial anomeric hydroxyl protons are shifted less than corresponding equatorial protons. Proton exchange with water is strongly acid and base catalyzed, but, at least in some cases, there seems to be an additional pH-independent mechanism involved. From the temperature effect on the shifts, and the effect of added dimethyl sulfoxide, we conclude that each hydroxyl group is bonded on average to two water molecules. This estimate of the hydration number for monosaccharides is far greater than those previously deduced from relaxation studies. It is suggested that the source of this difference lies in the residence times of the bound water molecules. Shifts of the hydroxyl proton resonances for sugars in methanol are compared with those for aqueous solutions and are found to be very similar. Hence it is concluded that these shifts do not reveal any special effects due to water structure. There are quite marked differences in the shifts for different sugars, and, in particular, the anomeric hydroxyl proton shifts for ketoses are smaller than those for aldoses.

Free radical activity following contraction-induced injury to the extensor digitorum longus muscles of rats.

The purpose of the study was to investigate the role of free radicals in the injury induced by a protocol of repeated pliometric (lengthening) contractions to the extensor digitorum longus (EDL) muscle in situ in rats. Previous data have indicated that prior treatment with the antioxidant polyethylene glycol-superoxide dismutase reduced the damage that was apparent at 3 days following this type of exercise. Three hours and 3 days following the protocol, the magnitude of the semiquinone-derived free radical signal observed by electron spin resonance spectroscopy (ESR) was not different for exercised and non-exercised skeletal muscles. A reduction in the protein thiol content of muscle was evident at 3 h, and was still apparent at 3 days. Three hours after the protocol, the total muscle glutathione content and the percentage in the oxidized form were unchanged, but by 3 days the percentage of muscle glutathione present in the oxidized form was elevated. The susceptibility of muscle to lipid peroxidation in vitro was reduced 3 days after the pliometric contractions. These data indicate that oxidation of protein thiols and glutathione may be involved in the secondary damage following pliometric contractions, but provide no evidence that the species involved were derived from mitochondrial semiquinone radicals.

A radiofrequency ESR spectrometer for in vivo imaging

Abstract A radiofrequency ESR spectrometer suitable for in vivo studies is described. The spectrometer utilizes a large loop-gap resonator, 10 cm in diameter and 15 cm in length, with an active volume of about 300 ml. This can be tuned to resonate at exactly 300 MHz for a wide range of samples and enables a fixed frequency source with high spectral purity to be used, minimizing noise demodulation. Automatic tuning, coupling, and phase control systems have been incorporated to compensate for motional artifacts in subsequent experiments using animals. Sensitivity is compared to that obtained using an aqueous cell at X band. For aqueous nitroxides, the minimum detectable concentration is about 2 × 10−7 M (sample volume - 200 ml, time constant = 0.01 s). The effects of physiological salt solution on sensitivity and RE depth penetration are reported and discussed. Coils for producing magnetic field gradients in three dimensions are also described and preliminary imaging results are shown.

On the electron spin resonance detection of RS radicals in irradiated solids: radicals of type RSSR–, RS–SR2, and R2SSR2+

It is suggested that radicals previously identified as RS, having g tensor components in the 2·002, 2·025, and 2·060 regions are better described as RS–SR2σ* radicals, structurally similar to the established radical anions, RS–SR–. Furthermore, a radical in irradiated thiodiglycolic acid previously thought to be (HO2CCH2)2S+ is almost certainly the dimer-cation (HO2CCH2)2S–S(CH2CO2H 2)+. The type of spectrum expected for RS radicals including HS is discussed, together with possible assignments.

Proton magnetic resonance study of the hydration of glucose

THE solvation of sugars by water has not been extensively studied in spite of its biochemical importance. Relaxation rates for 17O in monosaccharides have been used to study solvation1,2, the revised2 estimate of the solvation number being 5±1 water molecules for D-glucose and 2.5±1 for D-ribose at 5 °C, in agreement with 6±1 and 2.5±1 (respectively) derived from dielectric relaxation amplitudes. Again, compressibility data for aqueous sugars have been used3 to derive solvation numbers for monosaccharides, with results ranging from 2.5 to 4.0 at 25 °C. One limiting factor in nuclear magnetic resonance (NMR) studies of aqueous sugars is that the rate of proton exchange is fast and no separate resonances from the hydroxyl protons appear. We now report that at low temperatures the exchange rate becomes sufficiently slow in the pH range 4 to 8 to enable us to detect resolved resonances from the individual hydroxyl protons. The results are interpreted in terms of strong hydrogen bonding between water and glucose, the effective solvation number being at least 10.

Electron spin and electron nuclear double resonance of the [FeO2]− centre from irradiated oxyhemo- and oxymyoglobin

Abstract Exposure of glassy and icy solutions of oxyhemoglobin (HbO 2 ) and oxymyoglobin (MbO 2 ) to X- or ultraviolet irradiation at 77 K yields ESR- and ENDOR-active [FeO 2 ] − centres due to addition of an electron to the dioxygen. The centres are shown to be primary ones within the temperature range 4.2 K to 77 K. The rhombic ESR spectra exhibit distinct spectral changes due to various centres formed in α- and β-chains of HbO 2 and MbO 2 upon annealing. Depending on the solvent and pH, up to seven different centres can be distinguished in the ESR spectra, via shifts in g -elements in HbO 2 , and up to three in MbO 2 , all of which decay into ferric signals at about 200 K. Their spectral parameters, including 17 O-hyperfine splitting for the primary species, are given. ENDOR spectra reveal the presence of an exchangeable 1 H-coupling (approx. 10 MHz) in Mb and in both α- and β-chains of Hb for the primary centres which is attributable to an H-bond of the O 2 ligand to nitrogen NE2 of distal His-E7. Tentative assignments for several 1 H-couplings to amino acid residues (Val-E11, Phe-CD1, His-E7, His-F8) on the proximal and distal side of the heme are given for Mb. Differences between the various primary and the annealed centres mainly involve the loss of the exchangeable proton in Hb, Mb and isolated β-chains. The results are discussed in terms of an initial electron trap in the Fe-O-O unit stabilized by the hydrogen bond to the distal histidine proton, the latter being possibly transferred upon annealing.