CHROMOPHORE-ASSISTED LASER INACTIVATION OF PROTEINS IS MEDIATED BY THE PHOTOGENERATION OF FREE-RADICALS

Chromophore-assisted laser inactivation (CALI) is a technique that sel ectively inactivates proteins of interest to elucidate their in vivo f unctions. This method has application to a wide array of biological qu estions and an understanding of its mechanism is required for its judi cious application. We report here that CALI is not mediated by photoin duced thermal denaturation but by photogenerated free radicals. Therma l diffusion calculations suggest that the temperature changes resultin g from CALI are too small to cause thermal denaturation, and Arrhenius plots of CALI are inconsistent with a photothermal mechanism. CALI sh ows an energy dose reciprocity above a threshold and can be inhibited by free-radical quenchers, thus demonstrating a photochemical mechanis m of protein inactivation. The type of quenchers that are effective in inhibiting CALI indicates that the active species is a hydrogen abstr actor which is not derived from molecular oxygen. We suggest that the active free-radical species is the hydroxyl radical and its very short lifetime explains the spatial specificity of CALI such that half-maxi mal damage is effected within 15 angstrom from the dye moiety and no s ignificant damage occurs at 34 angstrom. The data are consistent with free-radical formation resulting from a sequential two-photon process.