Abstract

Singlet oxygen is produced by light-dependent excitation of molecular oxygen using photosensitizers. Using blue light and riboflavin (RF) or riboflavin phosphate (RFP) as photosensitizers, we studied the reaction of singlet oxygen with tertiary amines in aqueous solution because many are used as biochemical buffers and as electron donors in photoreduction reactions. The reactions of singlet oxygen with multiple tertiary amines, including ethylenediamine tetraacetic acid (EDTA), bicine, and triethanolamine (TEOA), produced micromolar hydrogen peroxide (H2O2) as the stable end product. For bicine and TEOA, but not EDTA, H2O2 yield increased as pH increased due to their higher amine pKa values. A white LED used in conjunction with riboflavin and tertiary amines also produced H2O2, a contaminant likely to form during tissue culture manipulations. Direct photoreduction of 2,6-dichlorophenolindophenol and 2,3-dimethoxy-5-methyl-p-benzoquinone was achieved using blue light, RF or RFP, and tertiary amines as electron donors. With RF, EDTA was the optimal electron donor for both substrates, whereas with RFP, bicine and TEOA were superior to EDTA. Photochemical redox cycling of both quinones produced H2O2 via singlet oxygen-dependent re-oxidation of reduced quinols. Several amine buffers, including HEPES and PIPES, reacted with singlet oxygen to produce H2O2 but did not function as electron donors in quinone photoreduction assays.

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