期刊
MOLECULAR PHARMACEUTICS
卷 16, 期 1, 页码 258-272出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.molpharmaceut.8b00979
关键词
photostability; monoclonal antibody; tyrosine; protein fragmentation; mass spectrometry
资金
- Genentch Foundation
Immunoglobulin gamma (IgG) monoclonal antibodies (mAbs) are glycoproteins that have emerged as powerful and promising protein therapeutics. During the process of production, storage and transportation, exposure to ambient light is inevitable, which can cause protein physical and chemical degradation. For mechanistic studies of photodegradation, we have exposed IgG4-Fc to UV light. The photoirradiation of IgG4-Fc with monochromatic UVC light at lambda = 254 nm and UVB light with lambda(max) = 305 nm in air-saturated solutions revealed multiple photoproducts originating from tyrosine side chain fragmentation at Tyr(300), Tyr(373), and Tyr(436). Tyr side chain fragmentation yielded either Gly or various backbone cleavage products, including glyoxal amide derivatives. A mechanism is proposed involving intermediate Tyr radical cation formation, either through direct light absorption of Tyr or through electron transfer to an initial Trp radical cation, followed by elimination of quinone methide. Product formation showed either no (cleavage of Tyr(373)) or significant (cleavage of Tyr(436)) inverse product solvent isotope effects (SIEs), indicating a role for proton transfer in the cleavage mechanism of Tyr(436). The role of electron transfer in the cleavage of Tyr(436) was further investigated through mutation of an adjacent Trp(381). This is the first observation of a photoinduced Tyr side chain cleavage reactions in a protein.
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