Abstract
Human eosinophils, stimulated with phorbol myristate acetate, were found to produce 1268 nm chemiluminescence characteristic of singlet oxygen. Singlet oxygen generation required the presence of bromide ion. A bromide ion concentration of 100 microM, comparable to the total bromine content of whole blood, was sufficient for the eosinophils to generate measurable amounts of singlet oxygen. For the conditions used (10(7) cells/ml and 10 micrograms/ml phorbol myristate acetate), the duration of the singlet oxygen generation was brief, about 5 min, and the total yield of singlet oxygen was modest, 1.0 +/- 0.1 microM. The cells remained viable after the singlet oxygen production ceased. This is the first demonstration of singlet oxygen production from living cells. The singlet oxygen generated by eosinophils likely results from a peroxidase-catalyzed mechanism, since a purified eosinophil peroxidase-hydrogen peroxide-bromide system was also shown to produce singlet oxygen. The unique properties of eosinophil peroxidase are illustrated by the fact that at p2H 7.0 and with 100 microM bromide, eosinophil peroxidase generated 20 +/- 2% of the theoretical yield of singlet oxygen, whereas under identical conditions, myeloperoxidase and lactoperoxidase produced only 1.0 +/- 0.1% and -0.1 +/- 0.1%, respectively.
Highlights
Human eosinophils, stimulated with phorbol myris- 13-14), a cell whose primary function is to destroy invasive tate acetate, were found to produce 1268 nm chemi- microbes
Our preliminary interest in the eosinophil peroxidase system as a potential source of singlet oxygen stemmed from reports that hypobromous acid-generating peroxidases could efficiently generate singlet oxygen if the enzymes were incubated with supraphysiological concentrations of bromide at an acid pH (6-8, 11-12)
Since eosinophils are able to efficiently oxidize physiological concentrations of bromide in thepresence of a 1000fold excess of chloride at neutralpH, the possibility that purified eosinophil peroxidase would have a similar predilection for bromide seemed likely
Summary
From the $Medical Service, Edward HinesJ, r. Since a purified eosinophil peroxidase-hydrogen peroxide-bromide system was shown to produce singlet oxygen. Recent studies have demonstrated that cell-free systems consisting of hydrogen peroxide, purified myeloperoxidase, and chloride can generate singlet oxygen, the yield was modest even under optimal conditions which included an acidic pH lower than that normally found within the phagosome (12). Weiss et al (21) demonstrated that human eosinophils triggered in a physiological mixof chloride and bromide (100 mMC1- and 20-100 p~ Br-) used eosinophil peroxidase, an enzyme distinct from myeloperoxidase, to preferentially oxidize bromide to a brominating intermediate with characteristics similar if not identical to hypobromous acid. Since other peroxidases efficiently generate singlet oxygen in thepresence of supraphysiological concentrations of bromide (6-8, 11-12), and since hydrogen peroxide is known to react with hypobromous acid at rates significantly faster than those for hypochlorous acid to generate singlet oxygen (20, 22), these authors postulated that.
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