Toward Understanding the Mechanism of Chromophore-assisted Laser Inactivation-Evidence for the Primary Photochemical Steps
ABSTRACT 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 appl...
Saved in:
Published in | Photochemistry and photobiology Vol. 81; no. 2; pp. 358 - 366 |
---|---|
Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
Oxford, UK
Blackwell Publishing Ltd
01.03.2005
|
Online Access | Get full text |
Cover
Loading…
Summary: | ABSTRACT
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. |
---|---|
Bibliography: | istex:BC3C888AC6923F5C164BC0E079F958AB34A72C6A Posted on the website on 28 December 2004 ark:/67375/WNG-SL3XN4H1-L ArticleID:PHP358 |
ISSN: | 0031-8655 1751-1097 |
DOI: | 10.1111/j.1751-1097.2005.tb00195.x |