Department of Pharmaceutical Chemistry , University of Kansas , 2095 Constant Avenue , Lawrence , Kansas 66047 , United States.
Mol Pharm. 2019 Jan 7;16(1):258-272. doi: 10.1021/acs.molpharmaceut.8b00979. Epub 2018 Dec 24.
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 λ = 254 nm and UVB light with λ = 305 nm in air-saturated solutions revealed multiple photoproducts originating from tyrosine side chain fragmentation at Tyr, Tyr, and Tyr. 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) or significant (cleavage of Tyr) inverse product solvent isotope effects (SIEs), indicating a role for proton transfer in the cleavage mechanism of Tyr. The role of electron transfer in the cleavage of Tyr was further investigated through mutation of an adjacent Trp. This is the first observation of a photoinduced Tyr side chain cleavage reactions in a protein.
免疫球蛋白 γ（IgG）单克隆抗体（mAbs）是糖蛋白，已成为强大且有前途的蛋白质治疗药物。在生产、储存和运输过程中，不可避免地会接触环境光，这会导致蛋白质物理和化学降解。为了研究光降解的机制，我们已经将 IgG4-Fc 暴露于紫外光下。在空气饱和溶液中，用单色 UVC 光（λ=254nm）和 UVB 光（λ=305nm）对 IgG4-Fc 的光辐照揭示了源自 Tyr、Tyr 和 Tyr 侧链断裂的多种光产物。Tyr 侧链断裂生成 Gly 或各种骨架断裂产物，包括乙二醛酰胺衍生物。提出了一种涉及 Tyr 自由基阳离子形成的中间步骤的机制，该过程可以通过 Tyr 的直接光吸收或通过初始 Trp 自由基阳离子的电子转移来实现，随后是醌亚甲基的消除。产物形成显示出无（Tyr 的断裂）或显著（Tyr 的断裂）的反产物溶剂同位素效应（SIE），表明质子转移在 Tyr 断裂机制中起作用。通过突变相邻的 Trp 进一步研究了电子转移在 Tyr 断裂中的作用。这是首次在蛋白质中观察到光诱导的 Tyr 侧链断裂反应。
