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Photoinduced Tyrosine Side Chain Fragmentation in IgG4-Fc: Mechanisms and Solvent Isotope Effects
Molecular Pharmaceutics ( IF 4.5 ) Pub Date : 2018-12-12 00:00:00 , DOI: 10.1021/acs.molpharmaceut.8b00979 Huan Kang 1 , Thomas J. Tolbert 1 , Christian Schöneich 1
Molecular Pharmaceutics ( IF 4.5 ) Pub Date : 2018-12-12 00:00:00 , DOI: 10.1021/acs.molpharmaceut.8b00979 Huan Kang 1 , Thomas J. Tolbert 1 , Christian Schöneich 1
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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 λmax = 305 nm in air-saturated solutions revealed multiple photoproducts originating from tyrosine side chain fragmentation at Tyr300, Tyr373, and Tyr436. 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 Tyr373) or significant (cleavage of Tyr436) inverse product solvent isotope effects (SIEs), indicating a role for proton transfer in the cleavage mechanism of Tyr436. The role of electron transfer in the cleavage of Tyr436 was further investigated through mutation of an adjacent Trp381. This is the first observation of a photoinduced Tyr side chain cleavage reactions in a protein.
中文翻译：
IgG4-Fc中的光诱导酪氨酸侧链断裂：机制和溶剂同位素效应
免疫球蛋白γ（IgG）单克隆抗体（mAb）是糖蛋白，已成为功能强大且有前途的蛋白质治疗剂。在生产，储存和运输过程中，不可避免地要暴露于环境光下，这可能导致蛋白质的物理和化学降解。为了进行光降解的机理研究，我们将IgG4-Fc暴露在紫外线下。在空气饱和溶液中分别用λ= 254 nm的单色UVC光和λmax = 305 nm的UVB光对IgG4-Fc进行光辐照，发现在酪氨酸300，酪氨酸373和酪氨酸436上酪氨酸侧链断裂产生了多种光产物。。Tyr侧链断裂产生Gly或各种主链裂解产物，包括乙二醛酰胺衍生物。提出了一种涉及中间Tyr自由基阳离子形成的机理，该机理是通过Tyr的直接光吸收或通过电子转移至初始的Trp自由基阳离子，然后消除醌甲基化物。产物形成显示无（Tyr 373的裂解）或显着（Tyr 436的裂解）逆产物溶剂同位素效应（SIEs），表明质子转移在Tyr 436的裂解机理中起作用。通过相邻Trp 381的突变，进一步研究了电子转移在Tyr 436裂解中的作用。。这是蛋白质中光诱导的Tyr侧链裂解反应的首次观察。
更新日期：2018-12-12
中文翻译：
IgG4-Fc中的光诱导酪氨酸侧链断裂：机制和溶剂同位素效应
免疫球蛋白γ（IgG）单克隆抗体（mAb）是糖蛋白，已成为功能强大且有前途的蛋白质治疗剂。在生产，储存和运输过程中，不可避免地要暴露于环境光下，这可能导致蛋白质的物理和化学降解。为了进行光降解的机理研究，我们将IgG4-Fc暴露在紫外线下。在空气饱和溶液中分别用λ= 254 nm的单色UVC光和λmax = 305 nm的UVB光对IgG4-Fc进行光辐照，发现在酪氨酸300，酪氨酸373和酪氨酸436上酪氨酸侧链断裂产生了多种光产物。。Tyr侧链断裂产生Gly或各种主链裂解产物，包括乙二醛酰胺衍生物。提出了一种涉及中间Tyr自由基阳离子形成的机理，该机理是通过Tyr的直接光吸收或通过电子转移至初始的Trp自由基阳离子，然后消除醌甲基化物。产物形成显示无（Tyr 373的裂解）或显着（Tyr 436的裂解）逆产物溶剂同位素效应（SIEs），表明质子转移在Tyr 436的裂解机理中起作用。通过相邻Trp 381的突变，进一步研究了电子转移在Tyr 436裂解中的作用。。这是蛋白质中光诱导的Tyr侧链裂解反应的首次观察。
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