Isolation and characterization of a monomethioninesulfoxide variant of interferon alpha-2b
Isolation and characterization of a monomethioninesulfoxide variant of interferon alpha-2b
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DOI：
10.1023/a:1016059902645
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发表时间：
1996-05-01
影响因子：
3.7
通讯作者：
Bausch, JN
中科院分区：
文献类型：
--
作者：
Gitlin, G;Tsarbopoulos, A;Bausch, JN
目的.分离和表征市售治疗性蛋白质干扰素α-2b的单甲硫氨酸亚砜变体。通过反相高效液相色谱法分离甲硫氨酸（Met）-氧化变体，并通过SDS-PAGE、肽图谱和胰蛋白酶/V8产生的肽片段的质谱分析进行表征。并对分离的变异株进行了生物学和免疫学活性评价。发现rHuIFN α-2b变体在rHuIFN α-2b分子的位置III含有Met亚砜残基。远紫外CD光谱显示α-螺旋含量的轻微损失和β-折叠贡献的增加。CD光谱表明，色谱条件和Met氧化都有助于观察到的二级结构变化。干扰素α-2b主要成分及其甲硫氨酸氧化变体对用于蛋白质免疫测定的单克隆抗体表现出不同的反应性。发现rHuIFN α-2b散装原料药溶液中存在单甲硫氨酸亚砜rHuIFN α-2b变体。通过比较胰蛋白酶/V8图谱和质谱分析，将Met（111）残基鉴定为Met亚砜。然而，Met（111）残基的氧化似乎对分子的生物活性没有可检测的影响。
Purpose. To isolate and characterize a monomethioninesulfoxide variant of the commercially available therapeutic protein interferon alpha-2b.Methods. The methionine (Met)-oxidized variant was isolated by reverse-phase high performance liquid chromatography and characterized by SDS-PAGE, peptide mapping and mass spectrometric analysis of the trypsin/V8-generated peptide fragments. The biological and immunological activities of the isolated variant were also evaluated.Results. The rHuIFN alpha-2b variant was found to contain a Met sulfoxide residue at position ill of the rHuIFN alpha-2b molecule. The far-UV CD spectra showed a slight loss of alpha-helical content and an increase in the beta-sheet contribution. The CD spectra indicate that both chromatographic conditions and Met oxidation contribute to the observed secondary structure changes. Both interferon alpha-2b main component and its methionine-oxidized variant showed different reactivity to monoclonal antibodies employed in immunoassays for the protein.Conclusions. A monomethioninesulfoxide rHuIFN alpha-2b variant was found to be present in the rHuIFN alpha-2b bulk drug substance in solution. The Met(111) residue was identified as Met sulfoxide by comparative tryptic/V8 mapping and mass spectrometric analysis. Nevertheless, the oxidation of the Met(111) residue did not seem to have a detectable effect on the biological activity of the molecule.