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1 March 2005 Toward Understanding the Mechanism of Chromophore-assisted Laser Inactivation—Evidence for the Primary Photochemical Steps
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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 β-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.

Elke Horstkotte, Torsten Schröder, Jens Niewöhner, Erwin Thiel, Daniel G. Jay, and Stefan W. Henning "Toward Understanding the Mechanism of Chromophore-assisted Laser Inactivation—Evidence for the Primary Photochemical Steps," Photochemistry and Photobiology 81(2), 358-366, (1 March 2005). https://doi.org/10.1562/2004-07-22-RA-240.1
Received: 22 July 2004; Accepted: 1 December 2004; Published: 1 March 2005
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