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Targeted Oxidation of Torpedo californica Acetylcholinesterase by Singlet Oxygen
Author(s) -
Weiner Lev,
Roth Esther,
Silman Israel
Publication year - 2010
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.1751-1097.2010.00857.x
Subject(s) - photosensitizer , chemistry , singlet oxygen , photochemistry , acetylcholinesterase , methylene blue , biophysics , fluorescence , phototoxicity , enzyme , biochemistry , oxygen , in vitro , organic chemistry , biology , physics , photocatalysis , quantum mechanics , catalysis
Abstract The photosensitizer, methylene blue (MB), is a strong reversible inhibitor of Torpedo californica acetylcholinesterase (AChE) in the dark. Under illumination it causes irreversible inactivation. Loss of fluorescence of the singlet oxygen ( 1 O 2 ) trap, 9,10‐dimethylanthracene, was retarded in the presence of AChE, and the rate of photo‐inactivation was increased in the presence of D 2 O, indicating that inactivation was due to 1 O 2 generated by the photosensitizer. CD revealed slightly reduced far‐UV ellipticity, and slightly enhanced binding of an amphiphilic probe, indicating limited unfolding of the photo‐oxidized AChE. However, both near‐UV ellipticity and intrinsic fluorescence were markedly reduced, suggesting photo‐oxidative damage to tryptophans, (Trp) supported by appearance of novel emission peaks ascribed to N ′‐formylkynurenine and/or kynurenine. Like other partially unfolded forms, the photo‐oxidized AChE was sensitive to proteolysis. Photosensitized inactivation produced exclusively chemically cross‐linked dimers, whereas irradiation of a partially unfolded state generated higher‐order oligomers. The active‐site gorge of AChE contains Trp in inhibitor‐binding sites that might be targets for photo‐oxidation. Indeed, reversible inhibitors retard photo‐inactivation, and photo‐inactivation destroys their binding sites. An excess of AChE protects paraoxonase from photo‐inactivation by sequestering the photosensitizer. Affinity photo‐oxidation of AChE by MB thus provides a valuable model for studying site‐specific photo‐inactivation of enzymes in both fundamental and clinical contexts.