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Hydroxyl radicals (OH.), generated by a phosphate-buffered Cu2+/H2O2 system, were detected by lucigenin-amplified chemiluminescence, deoxyribose degradation and benzoate hydroxylation. In each system the buffer, Hepes, was found to stimulate radical generation significantly. There are two main reasons for this effect: Hepes increases Cu2+ solubility in phosphate-buffered systems, and forms a complex with Cu2+ that is effective in generating OH. from H2O2. Pipes, a structurally similar buffer, and histidine, a known Cu2+ chelator, were found to have a similar effect. These data suggest that the crucial factor in such free-radical-generating systems is the availability of Cu2+, and that these actions of Hepes should be considered in the design of studies utilizing such systems.

Free-radical generation by copper ions and hydrogen peroxide. Stimulation by Hepes buffer