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Toward Understanding the Mechanism of Chromophore‐assisted Laser Inactivation–Evidence for the Primary Photochemical Steps ¶
Author(s) -
Horstkotte Elke,
Schröder Torsten,
Niewöhner Jens,
Thiel Erwin,
Jay Daniel G.,
Henning Stefan W.
Publication year - 2005
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.2005.tb00195.x
Subject(s) - chromophore , photobleaching , singlet oxygen , photochemistry , chemistry , fluorescence , singlet state , biophysics , oxygen , excited state , organic chemistry , optics , physics , nuclear physics , biology
Chromophore‐assisted laser inactivation (CALI) is a lightmediated 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.