Characterization of A2E free radicals

To characterize free radical forms of A2E that may be generated during its photooxidation and redox transformations. Methods: A2E (0.01 mM) was solubilized in pH 7.2, 10 mM phosphate buffer with 2%Triton X–100. Semi–reduced and semi–oxidized A2E species were obtained by interaction of the solubilized A2E with one–electron reducing and oxidizing radicals, respectively, generated by water radiolysis in the presence of selected scavengers of the primary radicals. The pulse radiolysis experiments were carried out using the Daresbury Laboratory Free Radical Research Facility. Results: An efficient interaction of the positively charged A2E molecule with the reducing formate radical was detected with the corresponding rate constant of about 109 M–1s–1. The resulting neutral radical (A2E.) exhibited strong abosorption in the near infrared and visible region with apparent maximum at 530 nm. Under the experimental conditions used, A2E. was a long–lived species decaying over hundreds of milliseconds to an unidentified product. However, in air saturated samples, A2E. interacted with oxygen (rate constant 3x108 M–1s–1) regenerating A2E and forming superoxide anion. A2E could also be converted to a cation radical (A2E.+) by interaction with the oxidizing bromide radical. The semi–oxidized A2E was formed with the rate constant of about 109 M–1s–1 and showed distinct absorption in the visible region with maximum at 590 nm. A2E.+ was a relatively short–lived species decaying over hundreds of microseconds to a secondary product that absorbed light in the region 500 – 600 nm. Conclusions: Two radical forms of A2E, namely A2E. and A2E.+, have been characterized spectrophotometrically and kinetically, providing basis for future study of free radical processes that might be responsible for the reported phototoxicity of this pyridinium bis–retionoid.