Singlet Oxygen Formation by a Peroxidase, H₂O₂ and Halide System

Abstract

Evidence for singlet oxygen formation has been obtained for the lactoperoxidase, H 2 0 2 and bromide system by monitoring 2,3-diphenyIfuran and diphenylisobenzofuran oxidation, 0 2 evolution, and chemiluminescence. This could provide an explanation for the cytotoxic and microbicidal activity of peroxidases and polymorphonuclear leukocytes. Evidence for singlet oxygen formation included the following. (a) Chemiluminescence accompanying the enzymic reaction was doubled in a deuterated buffer and inhibited by singlet oxygen traps. (b) The singlet oxygen traps, diphenylfuran and diphenylisobenzofuran, were oxidized to their known singlet oxygen oxidation products in the presence of lactoperoxidase, hydrogen peroxide and bromide. (c) The rate of oxidation of diphenylfuran and diphenylisobenzofuran was inhibited when monitored in the presence of known singlet oxygen traps or quenchers. (d) Oxygen evolution from the enzymic reaction was inhibited by singlet oxygen traps but not by singlet oxygen quenchers. (e) The traps or quenchers which were effective inhibitors in the experiments above did not inhibit peroxidase activity, were not competitive peroxidase substrates and did not react with the hypobromite intermediate since they did not inhibit hydrogen peroxide consumption by the enzyme.Using these criteria, various biological molecules were tested for their reactivity with singlet oxygen. Furthermore, by studying their effect on oxygen release by the enzymic reaction, it could be ascertained whether they were acting as singlet oxygen traps or quenchers.The microbicidal activity of myeloperoxidase or lactoperoxidase, hydrogen peroxide, and halides has been described by Klebanoff [I -31. These peroxidase systems have aIso been shown to be cytotoxic to tumor cells [4,5] as well as a variety of bacterial, fungal and viral species. The nature of the toxic component of these systems has not been determined but mounting evidence points to singlet oxygen ('02*) involvement. The microbicidal [6] and cytotoxic [7] property of chemically generated ' 0 2 * has been established. Krinsky has found that a carotenoidless mutant organism was killed rapidly by polymorphonuclear leukocytes, unlike the wild-type strain containing the '02* quencing carotenoids [S]. Direct evidence for '02* involvement has come from experiments by Allen and coworkers where it was shown that phagocytizing polymorphonuclear leukocytes [9] and isolated myeloperoxidase [ l o ] in the presence of H202 and C1-, emitted light which could be due to the relaxation of excited singlet oxygen to its ground state (triplet oxygen). Klebanoff has been able to inhibit the micro-____ bicidal activity of phagocytizing polymorphonuclear leukocytes using a '02* trap [6]. In a preliminary communication we have also demonstrated that the lactoperoxidase system can convert 2,5-diphenylfuran to its specific singlet oxygen product, cis-dibenzoyl ethylene [I I].Inhibition of this conversion using ' 0 2 * quenchers, and stimulation of conversion in 'H20 buffer (in which the half-l...

Sequential Injection Analysis with Chemiluminescence Detection for the Antioxidative Activity against Singlet Oxygen

“…Using a 100 mM sodium acetate buffer (pH 4.5), which has been employed in the batch method, [11][12][13] as a carrier solution, we investigated the effects of flow-rate of the carrier solution, the aspiration order and the concentrations of reagents on the CL intensity. The CL intensity obtained from DMSO (blank) was used as the index for the optimization of SIA-CL conditions.…”

“…The antioxidative activities of phenolic antioxidants, 11,12 fluvastatin and its metabolites, 13 and rosemary extract, 14 were successfully evaluated by the luminol CL methods. However, these methods based on batch technique are relatively laborious, time-consuming and wasteful of sample and reagents.…”

“…[11][12][13] These methods are based on the measurements of decreased amounts of 1 O2 by antioxidants. Among these methods, CL method has been widely used because of its high sensitivity and the use of inexpensive instruments.…”

“…16 In this paper, a SIA with CL detection for the measurement of antioxidative activity against 1 O2 was developed. Lactoperoxidase (LPO)-hydrogen peroxide (H2O2)-bromide ion system was used for the generation of 1 O2, [11][12][13][14] and the attenuation of luminol CL due to quenching of 1 O2 by each antioxidant was measured. The SIA-CL system was optimized with respect to the flow-rate of the carrier, the concentration of reagents and their aspiration order.…”

“…Several methods using electron spin resonance (ESR), [5][6][7] phosphorescence [8][9][10] or chemiluminescence (CL) detection [11][12][13] have been reported for antioxidative activity against 1 O2 which is generated photochemically, 5,6,[8][9][10] chemically 7 or enzymatically. [11][12][13] These methods are based on the measurements of decreased amounts of 1 O2 by antioxidants.…”