title: Role of surface exposed tryptophan as substrate generators for the antibody catalyzed water oxidation pathway.
abstract: The reaction of singlet oxygen with water to form hydrogen peroxide was catalyzed by antibodies and has been termed as the antibody catalyzed water oxidation pathway (ACWOP) (Nieva and Wentworth, Trends Biochem. Sci. 2004, 29, 274-278; Nieva et al. Immunol. Lett. 2006, 103, 33-38). While conserved and buried tryptophans in the antibody are thought to play a major role in this pathway, our studies with a monoclonal antibody, mAb-1 and its mutant W53A, clearly demonstrate the role of surface-exposed tryptophans in production of hydrogen peroxide, via the photo-oxidation pathway. Reactive oxygen species (ROS) such as singlet oxygen and superoxide were detected and site-specific tryptophan (Trp53) oxidation was observed under these conditions using RP-HPLC and mass spectrometry. The single mutant of the surface exposed Trp53 to Ala53 (W53A) results in a 50% reduction in hydrogen peroxide generated under these conditions, indicating that surface exposed tryptophans are highly efficient in transferring light energy to oxygen and contribute significantly to ROS generation. ACWOP potentially leads to the chemical instability of mAb-1 via the generation of ROS and is important to consider during clinical and pharmaceutical development of mAbs.
authors.affiliation: Late Stage Pharmaceutical Development, Genentech, Inc, South San Francisco, California 94080, United States. sreedhaa@gene.com
authors.forename: Alavattam
authors.initials: A
authors.lastname: Sreedhara
authors.forename: Kimberly
authors.initials: K
authors.lastname: Lau
authors.forename: Charlene
authors.initials: C
authors.lastname: Li
authors.forename: Brian
authors.initials: B
authors.lastname: Hosken
authors.forename: Frank
authors.initials: F
authors.lastname: Macchi
authors.forename: Dejin
authors.initials: D
authors.lastname: Zhan
authors.forename: Amy
authors.initials: A
authors.lastname: Shen
authors.forename: Daniel
authors.initials: D
authors.lastname: Steinmann
authors.forename: Christian
authors.initials: C
authors.lastname: Schöneich
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Lentz
mesh_terms: D000818:Animals; D017151:Antibodies, Catalytic / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D006861:Hydrogen Peroxide / Q000378:metabolism; D008027:Light; D008958:Models, Molecular; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism; D026082:Singlet Oxygen / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism*; D014867:Water / Q000378:metabolism*
chemical_list: D017151:Antibodies, Catalytic; D000911:Antibodies, Monoclonal; D017382:Reactive Oxygen Species; D014867:Water; D013481:Superoxides; D026082:Singlet Oxygen; D014364:Tryptophan; D006861:Hydrogen Peroxide