J.V. Bannister

The production of free radicals during the autoxidation of cysteine and their effect on isolated rat hepatocytes

Autoxidizing cysteine has been shown to produce thiyl and hydroxyl radicals. Hydrogen peroxide increased the yield of both radicals which was inhibited by catalase but stimulated by copper/zinc superoxide dismutase. This effect is due to increased hydrogen peroxide production by copper/zinc superoxide dismutase as a result of superoxide dismutation. The production of superoxide radicals could not be detected probably because of its low reactivity, however, measurement of oxygen uptake and reduction of ferricytochrome c by autoxidizing cysteine clearly implicate the involvement of super oxide radicals. The production of hydroxyl radicals is postulated to proceed through a fenton reaction, however, this may not necessarily be metal ion controlled. Autoxidizing cysteine disrupts the integrity of hepatocytes causing release of glutathione, adenosine triphosphate and lactate dehydrogenase indicating that it is of little use as a therapeutic agent.

Enhanced production of hydroxyl radicals by the xanthine-xanthine oxidase reaction in the presence of lactoferrin

The generation of hydroxyl radicals by the xanthine-xanthine oxidase reaction (C. Beauchamp and I. Fridovich (1970) J. Biol. Chem. 245, 4641-1616) has been shown to be increased by iron-saturated lactoferrin isolated from pig neutrophils. Hydroxyl radical production, measured by EPR spin trapping and by ethylene production from alpha-keto-gamma-methiol butyric acid, has been demonstrated to be produced by a Fenton-type Haber-Weiss reaction catalysed by lactoferrin. The possibility that lactoferrin catalyses such a reaction in vivo is considered.

A mechanism for primaquine mediated oxidation of NADPH in red blood cells

The incubation of NADPH with primaquine results in the formation of free radicals which were demonstrated by the electron spin resonance (ESR) technique of spin trapping using 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) as the spin trap. The free radicals formed were identified as the superoxide (DMPO-OOH) and hydroxyl (DMPO-OH) spin adducts of DMPO. Copper/zinc superoxide dismutase inhibited the formation of DMPO-OOH while it only partly inhibited the formation of DMPO-OH which could be totally inhibited by catalase. This indicates that the formation of hydroxyl radicals is not totally arising from the Haber-Weiss reaction. However since the formation of hydroxyl radicals is dependent on hydrogen peroxide, a non-metal catalysed reduction of hydrogen peroxide is postulated for their formation. Oxygen consumption during the reaction between primaquine and NADPH was found to be consistent with the spin trapping experiments and the rate of production of DMPO-OH indicates the formation of 1:1 catalytic complex between the two reactants. Quenching of the fluorescence of NADPH at 460 nm in the presence of primaquine indicates the formation of a charge transfer complex. When red blood cells are incubated with primaquine a hydroxyl spin adduct (DMPO-OH) is observed. The formation of this radical is probably the main cause of primaquine mediated toxicity.

THE COMPLETE AMINO ACID SEQUENCE OF HUMAN Cu/Zn SUPEROXIDE DISMUTASE

D. BARRA, F. MARTINI, J. V. BANNISTER*, M. E. SCHININP;, G. ROTILIO, W. I-I. BANNISTER+ and F. BOSSA Istituto di Chimica Biologica e Centro di Biologia Molecolare de1 CNR, University of Rome, Rome, Italy, *Inorganic Chemistry Department, University of Oxford, Oxford OXI 3QR, England and +Nuffield Department of Clinical Biochemistry, Radcliffe Infirmary, University of Oxford, Oxford OX2 6HE, England

Does caeruloplasmin dismute superoxide? No

Caeruloplasmin (ferroxidase, EC 1 .16.3 .l) is a copper containingplasmaa&ycoprotein.Three biological functions have been ascribed to caeruloplasmiri: (a) ferroxidase activity; (b) copper transport and Storage; and (c) maintenance of copper homeostasis in;the tissues. In [l] another function for caeruloplasmin has been proposed. These workers have demohstrated that caeruloplasmin inhibits the reduction of ferricytochrome c and of nitroblue tetrazolium by superoxide produced by the aerobic action of xanthine oxidase on hypoxanthine. Consequently it was proposed that caeruloplasmin may perform the function of scavenging any superoxide that leaks in the plasma where the levels of superoxide are extremely low. Here we report that caeruloplasmin does not catalyse the disproportionation of superoxide anion radicals generated by pulse radiolysis. However, the reduction of the type 1 copper(I1) in caeruloplasmin is observed.

The production of hydroxyl radicals by adriamycin in red blood cells

Spin trapping of the free radicals formed from the interaction between adriamycin and red blood cells resulted in the formation of a hydroxyl spin adduct. The formation of hydroxyl radicals was found to be inhibited by mannitol. Hemoglobin was found not to be obligatory for the formation of hydroxyl radicals which probably result from the reduction of hydrogen peroxide by adriamycin semiquinone.

The generation of hydroxyl radicals following superoxide production by neutrophil NADPH oxidase

Stimulated neutrophils generate superoxide and hydroxyl radicals. A membrane‐bound NADPH oxidase, inactive in the resting state, is responsible for superoxide production. The production of hydroxyl radicals is through a secondary reaction. A Fenton‐catalysed Haber—Weiss reaction is proposed. Transferrin was used as the catalyst in this investigation.

Isolation from neutrophil membranes of a complex containing active NADPH oxidase and cytochrome b-245

NADPH-dependent O2- forming activity was extracted with deoxycholate from subcellular particles of guinea-pig neutrophils following stimulation with phorbol myristate acetate. The solubilized enzyme was purified by chromatography on Ultrogel AcA22, by isopycnic glycerol density gradient centrifugation and by treatment with 0.4 M NaCl. This procedure yielded a high-molecular-weight complex containing phospholipids, cytochrome b-245 and NADPH oxidase activity. Cytochrome b was found to be purified to the same extent as NADPH oxidase activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the various purification fractions showed a progressive enrichment of a band whose molecular weight is 3.2 X 10(4). The enrichment of this protein band paralleled those of NADPH oxidase activity and of cytochrome b, indicating that it is a component of the oxidase system. The possibility that this band corresponds to either cytochrome b or a flavoprotein/cytochrome b complex is considered.

The formation of active oxygen species following activation of 1-naphthol, 1,2- and 1,4-naphthoquinone by rat liver microsomes

The hepatic microsomal metabolism of 1-naphthol, 1,2- and 1,4-naphthoquinone has been shown to generate active oxygen species by using electron spin resonance spin-trapping techniques. 1-Naphthol, in the presence of NADPH, and 1,2- and 1,4-naphthoquinone, with either NADH or NADPH, caused a stimulation in both the rate of microsomal oxygen consumption and the formation of superoxide spin adduct, 5,5-dimethyl-2-hydroxyperoxypyrrolidino-1-oxyl (DMPO-OOH). Superoxide dismutase, but not catalase, prevented the formation of this spin adduct, further supporting the suggestion that the superoxide free radical was the major oxy-radical formed during the microsomal metabolism of 1-naphthol and the naphthoquinones. These results are compatible with the suggestion that 1-naphthol may exert its toxicity to isolated hepatocytes and other cellular systems by metabolism to naphthoquinones followed by their redox cycling with concomittant generation of active oxygen species in particular superoxide free radicals.

The NADPH oxidase of guinea pig polymorphonuclear leucocytes

SummaryNADPH oxidase from stimulated guinea pig granulocytes was extracted with deoxycholate. The solubilized enzyme was stable in 20% glycerol. Solubilized enzyme was free of myeloperoxidase activity. The properties of the deoxycholate solubilized enzyme indicated that it is a high molecular weight complex with a flavoprotein, calmodulin and cytochrome b possibly forming part of the complex. Maximum activity was between pH 7.0 and 7.5. The Km value was 15.8 µM for NADPH and 434 µM for NADH indicating that NADPH is the preferential substrate.