Dioxygen-derived radicals in biological systems

Three instances of the involvement of dioxygen-derived radicals in biological systems are considered. The first concerns the formation of radicals in the haemolytic reactions induced by treatment of erythrocytes by phenylhydrazine, as an example of the so-called 'oxidant drugs'. The evidence for the formation of phenyl radicals is considered and their origin in the oxidation of phenylhydrazine by a ferryl derivative of haemoglobin postulated. The relevance to the formation of phenylated iron and porphyrin species is described. It is suspected that many instances of oxidative damage to cellular systems result from the coincidence of unsequestered redox-active metal ions (particularly those of iron and copper), reductants, and dioxygen. As an example, the damage to hepatocytes, grown in a culture medium containing cysteine, is described. The formation of radical species derived from dioxygen during the respiratory burst associated with phagocytosis is discussed. A new electrochemical method of detecting the superoxide ion produced during the respiratory burst is described. Particular emphasis is placed on the relation between the production of radical species such as the hydroxyl radical and the superoxide ion, and the extent of phagocytosis.     

The generation and subsequent fate of glutathionyl radicals in biological systems

Horseradish peroxidase-catalyzed oxidation of p-phenetidine in the presence of either glutathione (GSH), cysteine, or N-acetylcysteine led to the production of the appropriate thioyl radical which could be observed using EPR spectroscopy in conjunction with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide. This confirms earlier work using acetaminophen (Ross, D., Albano, E., Nilsson, U., and Moldéus, P. (1984) Biochem. Biophys. Res. Commun. 125, 109-115). The further reactions of glutathionyl radicals (GS.), generated during horseradish peroxidase-catalyzed oxidation of p-phenetidine and acetaminophen in the presence of GSH, were investigated by following kinetics of oxygen uptake and oxidized glutathione (GSSG) formation. Oxygen uptake and GSSG generation were dependent on the concentration of GSH but above that which was required for maximal interaction with the primary amine or phenoxy radical generated during peroxidatic oxidation of p-phenetidine or acetaminophen, suggesting that a secondary GSH-dependent process was responsible for oxygen uptake and GSSG production. GSSG was the only product of thiol oxidation detected during peroxidatic oxidation of p-phenetidine or acetaminophen in the presence of GSH, but under nitrogen saturation conditions its production was reduced to 8 and 33% of the corresponding amounts obtained under aerobic conditions in the cases of p-phenetidine and acetaminophen, respectively. Nitrogen saturation conditions did not affect horseradish peroxidase-catalyzed metabolism. This shows that the main route of GSSG generation in such reactions is not by dimerization of GS. but via mechanism(s) involving oxygen consumption such as via GSSG-. or via GSOOH.     

A quantitative test for superoxide radicals produced in biological systems.

The preparation and properties of a partially succinoylated cytochrome c, suited for the detection of superoxide anion radicals in liver microsomes, is reported. By succinoylation of 45% of the primary amino groups of horse heart cytochrome c the activity towards solubilized NADPH--cytochrome P-450 reductase was diminished by 99% compared with native cytochrome c. The capacities of cytochrome b5 and cytochrome c oxidase to reduce the succinoylated ferricytochrome c and oxidize succinoylated ferrocytochrome c respectively were decreased to a similar extent. However, the bimolecular rate constant for the reduction of the partially succinoylated ferricytochrome c by O2-. was estimated to be one-tenth of the value for the reaction of O2-. with native ferricytochrome c a pH 7.7. On this basis the quantification of O2-. generated by NADPH-supplemented liver microsomes became possible. The initial rates of succinoylated ferricytochrome c reduction determined at various finite concentrations of the cytochrome c derivative can be extrapolated to obtain true rates of O2-. generation in a homogeneous system. The problems encountered in the quantitative determination of O2-. produced in biological membranes, e.g. microsomes, are discussed.     

EPR spin-trapping of protein radicals to investigate biological oxidative mechanisms

Summary. Presently, free radicals and oxidants are considered to mediate from signaling circuits involved in physiology and pathology to cell and tissue injury. The elucidation of these many inter-related processes requires a better understanding of cellular oxidative mechanisms many of which are mediated by protein radicals. Here, we will discuss the potentialities of EPR spin-trapping of protein radicals to unravel oxidative mechanisms. An overview of the methodology and its application to identify protein residues that are the target of specific oxidants, characterize emerging oxidants, and discriminate radical from non radical mechanisms will be presented. The examples are based on work developed in our laboratories but will be discussed in a broad scenario to emphasize that simple experiments can provide relevant insights into the biological reactivity of known and emerging biological oxidants and into signaling mechanisms.     

From protein networks to biological systems

A system-level understanding of any biological process requires a map of the relationships among the various molecules involved. Technologies to detect and predict protein interactions have begun to produce very large maps of protein interactions, some including most of an organism's proteins. These maps can be used to study how proteins work together to form molecular machines and regulatory pathways. They also provide a framework for constructing predictive models of how information and energy flow through biological networks. In many respects, protein interaction maps are an entrée into systems biology.     

Free radicals in iron-containing systems

All oxidative damage in biological systems arises ultimately from molecular oxygen. Molecular oxygen can scavenge carbon-centered free radicals to form organic peroxyl radicals and hence organic hydroperoxides. Molecular oxygen can also be reduced in two one-electron steps to hydrogen peroxide in which case superoxide anion is an intermediate; or it can be reduced enzymatically so that no superoxide is released. Organic hydroperoxides or hydrogen peroxide can diffuse through membranes whereas hydroxyl radicals or superoxide anion cannot. Chain reactions, initiated by chelated iron and peroxides, can cause tremendous damage. Chain carriers are chelated ferrous ion; hydroxyl radical .OH, or alkoxyl radical .OR, and superoxide anion O2-. or organic peroxyl radical RO2.. Of these free radicals .OH and RO2. appear to be most harmful. All of the biological molecules containing iron are potential donors of iron as a chain initiator and propagator. An attacking role for superoxide dismutase is proposed in the phagocytic process in which it may serve as an intermediate enzyme between NADPH oxidase and myeloperoxidase. The sequence of reactants is O2----O2-.----H2O2----HOCl.     

The growing relevance of biological ene reactions

The ene reaction involves the addition of an ‘ene’ to an ‘enophile.’ The retro-ene reaction is the reverse of the ene reaction. In recent years various biological molecules have been found to form covalent intermediates (ene-adducts) that might be the result of an ene reactions. Such adducts have been characterized or implicated for dihydropyridines and pyridininum cofactors derived from vitamin B3, such as the reduced and oxidized forms of nicotinamide adenine dinucleotide (NADH/NAD); flavin cofactors derived from vitamin B2, such as flavin adenine dinucleotide, FAD, and flavin mononucleotide, FMN; vitamin C; the oxime intermediate of nitric oxide synthase; tyrosine; and other biomolecules. Given the ubiquitous nature of these cofactors, it might be speculated that the formation of ene-adducts is a more common principle in biochemistry.     

Transforming cyanobacteria into bioreporters of biological relevance

Microbial bioreporters play an important role in environmental monitoring and ecotoxicology. Microorganisms that are genetically modified with reporter genes can be used in various formats to determine the bioavailability of chemicals and their effect on living organisms. Cyanobacteria are abundant in the photosynthetic biosphere and have considerable potential with regards to broadening bioreporter applications. Two recent studies described novel cyanobacterial reporters for the detection of environmental toxicants and iron availability.     

Direct linkage of EGF to its receptor: Characterization and biological relevance

A small portion of the ¹²⁵I-EGF that binds specifically to intact cells or isolated membranes from a variety of sources becomes directly and irreversibly linked to EGF receptors. This provides a simple technique for affinity labeling the EGF receptor. Membranes isolated from the human epidermoid carcinoma cell line A431, which posesses extraordinarily high numbers of EGF receptors, gave rise to three major direct linkage complexes of MW = 160,000, 145,000, and 115,000. The time course for formation of each is similar, showing that ¹²⁵I-EGF can form direct linkage complexes with several preexisting forms of the EGF receptor. The direct linkage of EGF to receptor is slow in comparison to ¹²⁵I-EGF binding, but both processes have similar susceptibilities to competition by unlabeled EGF. EGF was modified chemically with the amino site-specific reagent, N-hydroxysuccinimidyl biotin. The biotinyl-EGF had a reduced capacity to engage in direct linkage complex formation with no concomitant reduction in its ability to bind to EGF receptors. Since native and biotinyl EGF have identical abilities to stimulate the uptake of ³H-thymidine into DNA when incubated with cultured murine 3T3 cells, the direct linkage of EGF to its receptor does not appear to play an important role in EGF-stimulated mitogenesis.