Magnetic nanoparticles bound with glutathione (GSH) are useful for diagnostics, enzyme immobilization, and affinity precipitation by using the strong and specific interaction of GSH with glutathione S-transferase (GST)-fused proteins. Our studies revealed that GSH-bound magnetic nanoparticles could be obtained using the covalent bond linkage of GSH and nanoparticles to promote the stability of bound GSH. To yield this conjugate, superparamagnetic iron oxide nanoparticles (SPIONs) were prepared and modified using tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES), which introduced amino groups that were then activated with maleic anhydride (MA) for covalent binding of GSH. After MA was used to activate the amino-grafted SPION for 24 h, the yield of GSH conjugation increased over 4 days from 37 % to 74 % of the original amine density on the surface as the incubation of GSH with MA-activated SPION. These GSH-bound magnetic nanoparticles, designated as SPION@silica-GSH with approximately 103 nmol GSH/mg particles, were ready for coupling with GST-fused protein through the GSH-GST affinity interaction. A GST-tagged small fragment of ubiquitin-like-specific protease 1 (sfULP1) was used as the model protein for immobilization on SPION@silica-GSH. ULP1 is a small ubiquitin-like modifier (SUMO) protease. Results indicated that this immobilized GST-sfULP1 could retain 87 % ± 5 % enzyme activity of free protease before immobilization and could catalyze the cleavage of the SUMO-fused peptide (SUMO-GLP-1) to obtain glucagon-like peptide-1, a peptide hormone for type 2 diabetes therapy.

中文翻译：

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谷胱甘肽在磁性纳米颗粒上的共价结合：用于固定小片段泛素样特异性蛋白酶 1 的应用

通过使用 GSH 与谷胱甘肽 S 转移酶 (GST) 融合蛋白的强烈和特异性相互作用，与谷胱甘肽 (GSH) 结合的磁性纳米颗粒可用于诊断、酶固定和亲和沉淀。我们的研究表明，可以使用 GSH 和纳米颗粒的共价键连接来获得 GSH 结合的磁性纳米颗粒，以提高结合 GSH 的稳定性。为了产生这种结合物，使用四乙氧基硅烷 (TEOS) 和 3-氨基丙基三乙氧基硅烷 (APTES) 制备和改性超顺磁性氧化铁纳米粒子 (SPION)，它们引入了氨基，然后用马来酸酐 (MA) 激活以共价结合 GSH。MA用于激活氨基接枝的SPION 24小时后，随着 GSH 与 MA 激活的 SPION 孵育，GSH 缀合的产率在 4 天内从表面原始胺密度的 37% 增加到 74%。这些 GSH 结合的磁性纳米颗粒被命名为 SPION@silica-GSH，具有大约 103 nmol GSH/mg 颗粒，准备通过 GSH-GST 亲和相互作用与 GST 融合蛋白偶联。泛素样特异性蛋白酶 1 (sfULP1) 的 GST 标记小片段用作固定在 SPION@silica-GSH 上的模型蛋白。ULP1 是一种小的泛素样修饰剂 (SUMO) 蛋白酶。结果表明，这种固定化的 GST-sfULP1 在固定化之前可以保留 87%±5% 的游离蛋白酶的酶活性，并且可以催化 SUMO 融合肽 (SUMO-GLP-1) 的裂解以获得胰高血糖素样肽-1，一种用于 2 型糖尿病治疗的肽激素。