title: Toward understanding the mechanism of chromophore-assisted laser inactivation--evidence for the primary photochemical steps.
abstract: Chromophore-assisted laser inactivation (CALI) is a light-mediated technique used to selectively inactivate proteins of interest to elucidate their biological function. CALI has potential applications to a wide array of biological questions, and its efficiency allows for high-throughput application. A solid understanding of its underlying photochemical mechanism is still missing. In this study, we address the CALI mechanism using a simplified model system consisting of the enzyme beta-galactosidase as target protein and the common dye fluorescein. We demonstrate that protein photoinactivation is independent from dye photobleaching and provide evidence that the first singlet state of the chromophore is the relevant transient state for the initiation of CALI. Furthermore, the inactivation process was shown to be dependent on oxygen and likely to be based on photooxidation of the target protein via singlet oxygen. The simple model system used in this study may be further applied to identify and optimize other CALI chromophores.
authors.affiliation: Xerion Pharmaceuticals AG, Munich, Germany.
authors.forename: Elke
authors.initials: E
authors.lastname: Horstkotte
authors.forename: Torsten
authors.initials: T
authors.lastname: Schröder
authors.forename: Jens
authors.initials: J
authors.lastname: Niewöhner
authors.forename: Erwin
authors.initials: E
authors.lastname: Thiel
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Jay
authors.forename: Stefan W
authors.initials: SW
authors.lastname: Henning
mesh_terms: D000042:Absorption; D004396:Coloring Agents / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry / Q000528:radiation effects; D007834:Lasers; D008027:Light; D008956:Models, Chemical; D010100:Oxygen / Q000737:chemistry; D010777:Photochemistry; D026082:Singlet Oxygen / Q000528:radiation effects; D013997:Time Factors; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000528:radiation effects
chemical_list: D004396:Coloring Agents; D005452:Fluoresceins; D026082:Singlet Oxygen; D001616:beta-Galactosidase; D010100:Oxygen