Effect of PEG molecular weight and linking chemistry on the biological activity and thermal stability of PEGylated trypsin
Effect of PEG molecular weight and linking chemistry on the biological activity and thermal stability of PEGylated trypsin
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DOI：
10.1016/j.ijpharm.2008.01.016
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发表时间：
2008-06-05
影响因子：
5.8
通讯作者：
Wiwattanapatapee, Ruedeekorn
中科院分区：
文献类型：
--
作者：
Treethammathurot, Benjaporn;Ovartlarnporn, Chitchamai;Wiwattanapatapee, Ruedeekorn
聚乙二醇化蛋白质通常用作治疗剂，但很少对聚乙二醇分子量和连接化学对生物活性，特别是缀合蛋白质的热稳定性的影响进行系统研究。在这里，使用琥珀酸酐 (SA)、氰尿酰氯 (CC) 或甲苯磺酰氯 (TC) 制备活化的单甲氧基聚乙二醇 (mPEG)（分子量 1100、2000 和 5000 g/mol），并用于合成胰蛋白酶缀合物库。使用 N-苯甲酰基-L-精氨酸对硝基苯胺 (BAPNA) 作为底物比较天然胰蛋白酶和 mPEG 修饰的胰蛋白酶缀合物的酶活性 (K(M)、V(max) 和 K(cat)），并使用 BAPNA 和 N-α-苯甲酰基-L-精氨酸乙酯盐酸盐 (BAEE) 作为底物测定它们的热稳定性。 分别测量酰胺酶和酯酶活性的底物。还在40℃下检查了缀合物化学对胰蛋白酶自溶的影响。含有较高分子量的mPEG（5000 g/mol）的PEG-胰蛋白酶缀合物比游离胰蛋白酶更稳定，并且含有5000 g/mol CC-mPEG的缀合物具有最好的热稳定性。 (c) 2008 Elsevier B.V. 保留所有权利。
PEGylated proteins are routinely used as therapeutics, but systematic studies of the effect of PEG molecular weight and linking chemistry on the biological activity and particularly the thermal stability of the conjugated protein are rarely made. Here, activated monomethoxypolyethylene glycol (mPEG)s (Mw 1100, 2000 and 5000 g/mol) were prepared using succinic anhydride (SA), cyanuric chloride (CC) or tosyl chloride (TC) and used to synthesise a library of trypsin conjugates. The enzyme activity (K(M), V(max) and K(cat)) of native trypsin and the mPEG-modified trypsin conjugates was compared using N-benzoyl-L-arginine p-nitroanilide (BAPNA) as a substrate, and their thermal stability determined using both BAPNA and N-alpha-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) as substrates to measure amidase and esterase activity respectively. The effect of conjugate chemistry on trypsin autolysis was also examined at 40 degrees C. PEG-trypsin conjugates containing the higher molecular weight of mPEG (5000 g/mol) were more stable than free trypsin, and the conjugate containing CC-mPEG 5000 g/mol had the best thermal stability. (c) 2008 Elsevier B.V. All rights reserved.