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Biomedical subjects

M C Symons

Publications and source records attributed to M C Symons.

At least 37 records · Page 2Linked to original sources
PubMed1997

Electron movement through proteins and DNA.

Nature utilizes the phenomenon of single electron transfer very widely, especially in metallo-proteins. In systems when the metal donor (D) is well separated from the acceptor (A) by polypeptide chains, the transferring electron is presumed to be bonded, in part, by these chains, which may influence the pathway taken. This situation can be probed by radiolytic injection of electrons into proteins at low temperatures. One aim of this brief review is to consider how information derived from such radiolysis studies, and followed by ESR spectroscopy, may possibly impinge on studies of D-A systems. Electrons can also be injected into duplex DNA in this way, and the results are compared with those for proteins. They are also considered in the light of recent studies of D-A electron-transfer via a polynucleotide strand. It seems that such transfers are very efficient, and it is tentatively suggested that Nature may also use this conductivity in some as yet undiscovered systems.

DNA↗
PubMed1996

Radicals generated by bone cutting and fracture.

When bone fractures, a remarkably high yield of radicals is generated. By working at 77K, these are stable enough for detection and study. Major species have been identified by ESR (EPR) spectroscopy as alkyl peroxyl radicals and RCH2. radicals. Also, a species thought to be O.- radicals, formed in the mineral phase, was detected in relatively low yield. It is suggested that as a break is generated, the mineral crystallites separate at grain boundaries with no major chemical damage, but that the tightly bound collagen strands running through the mineral phase are forced to break homolytically. Some probably react together, others react with oxygen, while the remainder are trapped in their primary form. The characteristic hyperfine triplet is assigned in part to RCH2. radicals from glycine units, which constitute about one-third of the total amino acid components in collagen, and in part to RNHCO radicals. The possible significance of the formation of active radicals during bone fracture is discussed.

Animals↗
PubMed1996

When is a radical not a radical?

It is not commonly realised that symmetrical Fe(III) complexes are expected to have EPR spectra dominated by a symmetrical narrow line close to the free-spin g-value. The aim of this note is to stress that such features can be mistakenly identified as the spectra for stable organic radicals. This situation is a particular problem in studies of animal or plant tissue samples.

Animals↗
PubMed1996

Redox cycling of human methaemoglobin by H2O2 yields persistent ferryl iron and protein based radicals.

The formation and reactivity of ferryl haemoglobin (and myoglobin), which occurs on addition of H2O2, has been proposed as a mechanism contributing to oxidative stress associated with human diseases. However, relatively little is known of the reaction between hydrogen peroxide and human haemoglobin. We have studied the reaction between hydrogen peroxide and purified (catalase free) human metHbA. Addition of H2O2 resulted in production of both ferryl haem iron (detected by optical spectroscopy) and an associated protein radical (detected by EPR spectroscopy). Titrating metHbA with H2O2 showed that maximum ferryl levels could be obtained at a 1:1 stoichiometric ratio of haem to H2O2. No oxygen was evolved during the reaction, indicating that human metHbA does itself not possess catalytic activity. The protein radicals obtained in this reaction reached a steady state concentration, during hydrogen peroxide decomposition, but started to decay once the hydrogen peroxide had been completely exhausted. The presence of catalase, at concentrations around 10(4) fold lower than metHb, increased the apparent stoichiometry of the reaction to 1 mol metHb: approximately 20 mol H2O2 and abolished the protein radical steady state. The biological implications for these results are discussed.

Catalase↗
PubMed1995

Polyamines to target drugs to DNA.

In neutral solutions polyamines are fully protonated, and hence are really polyammonium cations (PAC). Spermine, for example, carries four positive charges in a linear system, H3N+(CH2)3N+H2(CH2)4N+H2 (CH2)3N+H3. There is a very powerful coulombic interaction between aqueous DNA and such cations, thus the cations are attracted to the DNA over large distances, and once close to the DNA normally remain there for long periods. A key issue is; are the cations mobile, or do they remain at one preferred site for significant periods? The latter is the currently preferred concept, but NMR and EPR evidence will be presented in favour of the former. If the former is correct, then PACs may be able to act as good drug delivery systems. In its simple form the concept is that any drug that acts directly on DNA can be chemically bound to a PAC. Once in the cell, this PAC-drug complex (PAC-D) will be carried to DNA and will move very rapidly along the exposed strands until it recognises the site of action. This may be some special base sequence region, a damaged site, or the PAC-D unit may simply be present prior to potential damage, so that this can be repaired very rapidly. Some of our current studies on these systems are described.

Biogenic Polyamines↗
PubMed1995

Failure of electron paramagnetic resonance spectroscopy studies to detect elevated free radical signals in liver biopsy specimens from patients with alcoholic liver disease.

Electron paramagnetic resonance spectroscopy (EPR) was used to study free radicals and transition metal complexes in liver tissue taken from patients with liver disease. Samples were frozen to 77K directly following biopsy to prevent deterioration. Our major aim was to compare signals from patients suffering from alcohol abuse with those from patients having liver damage not induced by alcohol. Samples were obtained from 19 chronic alcohol abusers and 7 non-alcoholic liver disease patients. Of the 19 alcoholic patients, 18 had an increased fat content, 6 had Mallory's hyaline, 12 had an acute inflammatory response, 9 had increased stainable iron and 4 had evidence of fibrosis. A signal derived from free radicals with a spectroscopic splitting factor of g = 2.0045 was found in all samples. This signal in the alcoholic patients had a mean amplitude of 2.96 cm (+/- 1.42 SD), and in patients with non-alcoholic liver disease 2.12 cm (+/- 0.82) (p = 0.10 NS), measured under identical instrument settings. The molar proportion of diene conjugated linoleic acid (DCLA), a free radical marker, in the sera of alcoholic patients was 2.68% (+/- 1.93), but did not correlate with the free radical signals obtained by EPR spectroscopy. Also, there was no correlation between the free radical derived EPR signal and fat content, Mallory's hyaline, inflammatory infiltrate, iron or fibrosis in the liver biopsy specimens. Similarly the concentrations of aspartate transaminase, albumin, and gamma-glutamyl transferase in serum samples showed no correlations with free radical concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

DNA in glasses at 77 K: high energy ionizing radiation versus UV electron ejection.

Most in the field of ionizing radiation damage to DNA in frozen aqueous solutions agree that two major types of radical ions are formed, i.e. G+/A+ and T-/C-. The main evidence stems from EPR and strand break studies. Fluid solutions exposed to laser light are known to give G+ and esolv- with low yields of single strand breaks. We have explored this contrast by photoionizing DNA solutions at 77 K, in the expectation that this would prevent the formation of esolv- and hence that the results might be similar to those for high energy radiation. They are not: the results show only the formation of G+ (or) (A+), the fate of the ejected electrons is unclear except for sodium perchlorate glasses when they react to give O-.

DNA↗
PubMed1994

Electron spin resonance studies of nitrosyl haemoglobin in human liver, colon and stomach tumour tissues.

Iron nitrosyl haemoglobin (HbFeNO) gives well defined ESR spectra, and can be detected at room temperature, in contrast with most transition metal complexes of biological importance. This is because the unpaired electron remains strongly localised on the NO ligand. It is of importance because it proves the formation of nitric oxide, which unfortunately cannot be detected directly by ESR spectroscopy. We have studied a range of tissues taken from human liver, colon and stomach tumours which have been directly frozen to 77K and studied at 77K. The results show that formation of HbFeNO is rare in tissue adjacent to tumour tissue ("peripheral tissue"), but is always found in necrotic central regions, if present. However, in several cases, HbFeNO was also detected in tumour tissue which was not necrotic. Two factors contribute to the formation of this complex. One is the presence of "free" NO molecules in the cellular regions, and the other is the presence of deoxyferrohaemoglobin, since neither ferrihaemoglobin nor oxyhaemoglobin react to give this complex. [For systems containing myoglobin these comments include the possibility of the formation of nitrosylmyoglobin, which gives very similar ESR spectra.

Adenocarcinoma↗
PubMed1994

The effect of myoglobin on the stability of the hydroxyl-radical adducts of 5,5 dimethyl-1-pyrolline-N-oxide (DMPO), 3,3,5,5 tetramethyl-1-pyrolline-N-oxide (TMPO) and 1-alpha-phenyl-tert-butyl nitrone (PBN) in the presence of hydrogen peroxide.

The hydroxyl radical adducts of 5,5 dimethyl-1-pyrolline-N-oxide (DMPO) and 3,3,5,5 tetramethyl-1-pyrolline-N-oxide (TMPO) formed in the presence of hydrogen peroxide and FeII are normally quite stable, but in the presence of 5-20 micromolar myoglobin their ESR signals decay rapidly. This decay probably reflects further oxidation of the adduct to nonparamagnetic products. The ESR signal of the hydroxyl radical adduct of 1-alpha-phenyl-tert-butyl nitrone (PBN) formed under similar conditions is subject to non-heme dependent attenuation, possibly via hydroxyl radical scavenging, but not to heme dependent decay. Hydrogen peroxide readily converts myoglobin to its ferryl (FeIV) derivative, and this centre may be responsible for the oxidation of the DMPO and TMPO adducts. The different behaviour of PBN may be due to differences in susceptibility to ferrylmyoglobin mediated oxidation, or to steric differences controlling access to the heme pocket of myoglobin, and is relevant to the choice of spin trap for biological experiments aimed at detecting hydroxyl radicals in the presence of myoglobin or other heme proteins.

Cyclic N-Oxides↗
PubMed1993

Electron paramagnetic resonance spectroscopy of stable free radicals in the liver compared with ultrastructural and functional damage in a rat model of alcohol- and iron-overload.

1. Electron paramagnetic resonance spectroscopy was used to study free-radical signals in freeze-clamped frozen liver tissue from rats after a 1 year period of dietary supplementation with alcohol, iron, or alcohol and iron. In alcohol-fed, iron-fed and alcohol- and iron-fed animals, mild histological damage was seen on light microscopy and evidence of mitochondrial and nuclear injury was identified by electron microscopy. 2. Subcellular fractionation studies showed an increase in the activity of the peroxisomal marker catalase (P < 0.01) in alcohol-fed rats compared with controls, but a fall of 82% (P < 0.001) in alcohol- and iron-fed animals. The activity of the mitochondrial marker succinate dehydrogenase rose by 7% (not significant) in alcohol-fed animals and by 17% (not significant) in iron-fed animals, but fell by 94% (P < 0.001) in alcohol- and iron-fed animals, suggesting serious impairment of mitochondrial function. 3. Iron overload was substantial in the iron-fed animals and there was an excellent correlation between liver iron concentration and iron-derived signals by electron paramagnetic resonance spectroscopy (P < 0.001). A clear free-radical signal of g = 2.003-2.005 was detected in all liver samples, but there was no significant difference in the magnitude of this signal in any study group. 4. The absence of any increase in the stable free-radical signal, even in the presence of considerable hepatic damage, does not support the hypothesis that free radicals mediate alcoholic liver disease in this animal model, although the results cannot be taken as proof against this hypothesis.

Animals↗
PubMed1993

Marked effect of buffers on yield of single- and double-strand breaks in DNA irradiated at room temperature and at 77 K.

We have shown that the protocol for handling plasmid (pBR 322) DNA for radiation studies, which normally involves using quite high concentrations of Tris or phosphate buffers, is equally satisfactory in the absence of added buffers provided samples are stored at 0 degree C, or used directly. On exposure of aqueous solutions at 77 K, Tris buffer acts as a weak protecting agent, but phosphate buffers at the same pH act as remarkably effective sensitizing agents, giving ca. 100-fold increases in strand breaks. At room temperature there is again a marked difference between the aqueous and buffered systems, but in this case both the Tris and the phosphate systems are protective. Possible reasons for these contrasting results are discussed, and the advantages of using simple aqueous solutions are stressed.

Buffers↗
PubMed1993

Interaction of ethanol with haemoglobin: implications for alcohol abuse.

Ingestion of large amounts of ethanol results in a variety of toxicological consequences of which reduced oxygen supply is a marked respiratory effect. Experimentation involving the in-vivo administration of ethanol to rats, identifies the formation of ferri-haemoprotein species and hydroxyl radicals in the liver. Our work on the in-vitro effect of ethanol on oxyhaemoglobin shows the formation of ferri-haemoglobin and the alpha-hydroxyethyl radical as an intermediate. The impairment of oxygen transport efficiency of haemoglobin may be a result of its oxidation to the non-functional ferrihaemoglobin form.

Alcoholism↗
PubMed1993

The effects of myoglobin and apomyoglobin on the formation and stability of the hydroxyl radical adduct of 5,5'-dimethyl-1-pyrroline-N-oxide.

When aqueous solutions of the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) are treated with hydrogen peroxide in the presence of either FeII or light, the hydroxyl radical adduct DMPO-OH is formed, with a characteristic 4 line ESR spectrum. When oxy- or metmyoglobin is added to such a system the initial yield and the halife of DMPO-OH are reduced, and at high myoglobin concentrations (about 0.1 mmol dm-3) DMPO-OH becomes undetectable. Using the stable nitroxide 2,2,6,6-tetramethyl-1-piperidinyloxy-N-oxyl (TMPO) for comparison it was found that neither hydrogen peroxide nor myoglobin alone caused a loss of signal, but together a marked loss of signal was induced. From the evidence of these and other experiments it was concluded that the DMPO-OH adduct reacts with hydrogen peroxide and myoglobin to give non-paramagnetic products, and hence that the use of the DMPO spin trap to detect hydroxyl or other active radicals in systems containing physiological concentrations of myoglobin may give misleading results.

Apoproteins↗
PubMed1992

Biological methane activation involves the intermediacy of carbon-centered radicals.

The spin-trapping technique has demonstrated that carbon-centered radicals are produced during soluble-methane-monooxygenase catalysis of the hydroxylation of several different types of substrate. The resulting spin-adducts were identified from the hyperfine splitting constants in their EPR spectra. Isotopic labelling showed unequivocally that the trapped radicals were derived from substrate. The carbon-centered substrate radicals are believed to result from hydrogen-atom abstraction by a ferryl species in a cytochrome-P-450-like mechanism. No hydroxy radical nor an oxygen-based radical of any kind was detected in any of the spin-trapping experiments.

Carbon↗
PubMed1989

Effect of ionizing radiation on haemoglobin: the oxy-derivative of haemoglobin Iwate.

It is well established that exposure of oxyhaemoglobin to ionizing radiation results in remarkably selective electron addition to the (FeO2) unit, giving a novel species, (FeO2)-, in which the extra electron is largely localized on iron and dioxygen. This work has now been extended to haemoglobin (Hb.) Iwate. The haemoglobin M. Iwate used is a mutant haemoglobin having only Fe(III) alpha-chains by oxy beta-chains (alpha 2 Met beta 2 oxy). The haem iron atoms in the alpha-chains are coordinated in the fifth site by a proximal tyrosine in place of histidine. This unit is a high-spin Fe(III) with axial symmetry and prominent electron spin resonance (ESR) features in the g = 6 and g = 2 regions. On exposure to 60Co gamma-rays at 77 K, efficient electron addition occurred at both types of iron centre, giving Fe(II) and (FeO2)- units. The former was monitored by the decrease of the g = 6 feature for Fe(III) and the latter by the growth of g-features at 2.254 (gx), 2.149 (gy) and 1.967 (gz). These values are close to those for the FeO2- centre formed in the beta-chains of normal oxyhaemoglobin. On annealing above 77 K, two changes occurred: first there was a small but clear increase in gx and gy, followed by a marked reduction in gx and gy giving g-values close to those for the centre formed directly in the alpha-chains of the normal protein. Finally, this intermediate species gave a centre having gx = 2.310, gy = 2.180 and gz = 1.935. These values are typical of low-spin Fe(III) haemoglobin and are assigned to the protonated complex, Fe(III)O2H. Ultimately at ca. room temperature, this was converted into the high-spin, met-form, with a gain in the g = 6 feature. These results established that the beta-chain centre in Hb. Iwate behave in the same way as isolated beta-chains. They also confirm that electron addition to the oxy-units is facile, even in the presence of Fe(III) units in each tetramer. The results also confirm that electron capture to give (FeO2)- units is not followed by internal electron-transfer to give Fe(II) from the Fe(III) centres in the alpha-chains.

Cobalt Radioisotopes↗