Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity
Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity
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DOI：
10.1016/j.enzmictec.2013.01.004
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发表时间：
2013-04-10
影响因子：
3.4
通讯作者：
McShane, Michael J.
中科院分区：
文献类型：
--
作者：
Ritter, Dustin W.;Roberts, Jason R.;McShane, Michael J.
研究了葡萄糖氧化酶在其糖基化位点的靶向PEG化，以确定对酶活性的影响，以及生物缀合物在光学生物传感测定中的潜力。甲氧基聚（乙二醇）-酰肼（4.5 kDa）共价偶联高碘酸氧化的糖基化位点的尼日尔曲霉葡萄糖氧化酶。使用凝胶电泳、液相色谱、质谱和动态光散射对生物缀合物进行表征。凝胶电泳数据显示，PEG化方案导致了细胞内蛋白质的急剧增加（约100%）。100 kDa）的蛋白质亚基的表观分子量，完全转化为生物缀合物;液相色谱数据证实了这种分子大小的大幅增加。质谱数据证明PEG化的程度为每个葡萄糖氧化酶二聚体六个聚（乙二醇）链。动态光散射数据表明在聚乙二醇化GOx样品中不存在高阶低聚物。为了评估稳定性，在不存在葡萄糖的情况下，以及在暴露于5% w/v葡萄糖24小时之前和之后，在29天的过程中的多个时间点进行了一式三份的酶活性测定。在95%的置信水平下，生物缀合物的性能在29天时间段内以及在24小时葡萄糖暴露后的活性保留方面在统计学上等同于天然葡萄糖氧化酶。最后，将生物缀合物包埋在含有氧敏感性磷光体的聚（甲基丙烯酸2-羟乙酯）水凝胶内，并且该构建体显示出在生理相关的葡萄糖范围（即，0-400 mg/dL）;据我们所知，这代表了聚乙二醇化葡萄糖氧化酶并入光学生物传感测定的首次证明。(C)2013 Elsevier Inc. All rights reserved.
Targeted PEGylation of glucose oxidase at its glycosylation sites was investigated to determine the effect on enzymatic activity, as well as the bioconjugate's potential in an optical biosensing assay. Methoxypoly(ethylene glycol)-hydrazide (4.5 kDa) was covalently coupled to periodate-oxidized glycosylation sites of glucose oxidase from Aspergillus niger. The bioconjugate was characterized using gel electrophoresis, liquid chromatography, mass spectrometry, and dynamic light scattering. Gel electrophoresis data showed that the PEGylation protocol resulted in a drastic increase (ca. 100 kDa) in the apparent molecular mass of the protein subunit, with complete conversion to the bioconjugate; liquid chromatography data corroborated this large increase in molecular size. Mass spectrometry data proved that the extent of PEGylation was six poly(ethylene glycol) chains per glucose oxidase dimer. Dynamic light scattering data indicated the absence of higher-order oligomers in the PEGylated GOx sample. To assess stability, enzymatic activity assays were performed in triplicate at multiple time points over the course of 29 days in the absence of glucose, as well as before and after exposure to 5% w/v glucose for 24h. At a confidence level of 95%, the bioconjugate's performance was statistically equivalent to native glucose oxidase in terms of activity retention over the 29 day time period, as well as following the 24h glucose exposure. Finally, the bioconjugate was entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct was shown to respond approximately linearly with a 220 +/- 73% signal change (n = 4,95% confidence interval) over the physiologically-relevant glucose range (i.e., 0-400 mg/dL); to our knowledge, this represents the first demonstration of PEGylated glucose oxidase incorporated into an optical biosensing assay. (C) 2013 Elsevier Inc. All rights reserved.