The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is frequently used to identify free radicals that are generated photochemically using dyes as photosensitizers. When oxygen is present in such systems, singlet oxygen (1O2) may be produced and can react with DMPO. We have studied the reaction of DMPO with 1O2 in aqueous solutions over a wide range of pH, using micellar Rose Bengal (pH 2−13) and anthrapyrazole (pH < 2) as photosensitizers. We found that DMPO quenches 1O2 phosphorescence (k q = 1.2 × 106 M-1 s-1), thereby initiating oxygen consumption that is slow at pH 10 but increases about 10-fold at pH < 6. This oxygen consumption is a composite process that includes efficient oxidation of both DMPO and its degradation products. The oxidation products include both products in which the DMPO pyrroline ring remains intact (DMPO/•OH and 5,5-dimethyl-2-oxo-pyrroline-1-oxyl (DMPOX) radicals) and those in which it becomes opened (nitro and nitroso products). The nitroso product itself strongly quenched 1O2 phosphorescence, while (photo)decomposition of the nitroso group, presumably to nitric oxide (NO•), produced nitrite as a minor product. We propose that 1O2 adds to the >CN(O) bond in DMPO, producing a biradical, >C(OO•)N•(O). This biradical may follow one of two pathways: (i) It may be protonated and rearrange to a strongly oxidizing nitronium-like moiety, which could be reduced to the DMPO hydroperoxide radical DMPO/•O2H while oxidizing another DMPO moiety to ultimately form DMPOX. The DMPO/•O2H could undergo further redox decomposition, e.g. via the known Fenton-like reaction, to produce both free •OH radical and the DMPO/•OH radical. (ii) The biradical >C(OO•)N•(O) may cyclize to a 1,2,3-trioxide (ozonide), which could open the pyrroline ring to form 4-methyl-4-nitropentan-1-al and 4-methyl-4-nitrosopentanoic acid. Because the oxidation of DMPO by 1O2 leads to both rapid O2 depletion and the formation of transients and products that might interfere with trapping and identification of free radicals, DMPO should be used with caution in systems where 1O2 is produced.
Singlet molecular oxygen (1O2) is one of the major agents responsible for (photo)oxidative damage in biological systems including human skin and eyes. It has been reported that the neural hormone melatonin (MLT) can abrogate 1O2-mediated cytotoxicity through its purported high antioxidant activity. We studied the interaction of MLT with 1O2 in deuterium oxide (D2O), acetonitrile and methanol by measuring the phosphorescence lifetime of 1O2 in the presence of MLT and related indoles for comparison. Rose bengal (RB) was used as the main 1O2 photosensitizer. The rate constant (kq) for the total (physical and chemical) quenching of 1O2 by MLT was determined to be 4.0 x 10(7) M(-1) s(-1) in D2O (pD 7), 6.0 x 10(7) M(-1) s(-1) in acetonitrile, and 6.1 x 10(7) M(-1) s(-1) in methanol-d1. The related indoles, tryptophan, 5-hydroxyindole, 5-methoxytryptamine, 5-hydroxytryptamine (5-OH-T, serotonin), 6-hydroxymelatonin (6-OH-MLT) and 6-chloromelatonin quenched 1O2 phosphorescence with similar kq values. We also compared the photosensitized photobleaching rate of MLT with that of other indoles, which revealed that MLT is the most sensitive to 1O2 bleaching. Hydroxylation of the indole moiety in 5-OH-T and 6-OH-MLT makes them more sensitive to photodegradation. In the absence of exogenous photosensitizers MLT itself can generate 1O2 with low quantum yield (0.1 in CH3CN) upon UV excitation. Thus, the processes we investigated may occur in the skin and eyes during physiological circadian rhythm (photo)signaling involving MLT and other indoles. Our results indicate that all the indoles studied, including MLT, are quite efficient yet very similar 1O2 quenchers. This directly shows that the exceptional antioxidant ability proposed for MLT is unsubstantiated when merely chemical mechanism(s) are considered in vivo, and it must predominantly involve humoral regulation that mobilizes other antioxidant defenses in living organisms.
The photooxidation of N,N-diethylhydroxylamine (DEHA) by Rose Bengal (RB) has been investigated in micellar and nonmicellar aqueous solutions. We measured the quantum yield of oxygen consumption forming H2O2 and monitored two intermediates, the superoxide and diethylnitroxide radicals. When the pH was varied, the quantum yield of oxidation remained constant for 6 < pH < 10.5, decreased in acidic pH, and increased considerably in NaOH solution; these changes could be attributed to the protonation and dissociation processes of the > N-OH moiety of DEHA. The formation of diethylnitroxide radical was enhanced by superoxide dismutase or strong alkaline solution. Around neutral pH, the oxidation proceeded mainly via electron transfer from DEHA to the RB triplet (kq = 10(7) M-1 s-1) with little 1O2 participation (kq < 10(5) M-1 s-1). However, when RB was incorporated into micelles in alkaline solution, the contribution of the singlet oxygen pathway increased at the expense of electron transfer, which was inhibited by the less polar micellar environment. Dark autoxidation of DEHA was accelerated by heavy metal impurities and increased very strongly in NaOH solution.
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