摘要
Hirudin, the most potent inhibitor of thrombin found in nature, has a short half-life in serum, which sig-nificantly limits its clinical application as an anticoagulant. Recently, PEGylation has been commonly used as an effective method to prolong its half-life in serum. In contrast to the nonspecific pEGylation under basic conditions that targets lysine residues randomly, PEGylation sites under mildly acidic conditions preferably targets histidine residues, and there is only one histidine residue at 51 in r-hirudin; therefore, succinimidyl carbonyl methoxy poly-ethylene glycol （SC-mPEG, 20000） was attached to r-hirudin at mildly acidic pH to favor the formation of mono-PEGylated r-hirudin. The reaction mixture with high mono-PEGylated ratio was easily separated by a one-step ion-exchange chromatographic （IEC） procedure. Approximately 79.71% of the mono-PEGylated r-hirudin was PEGylated at His51, which showed that the acidic PEGylation operation prevented the PEGylation of active center （Lys47） of r-hirudin in pdnciple. Mono-PEGylated.product with purity higher than. 95% was obtained as the pre-dominant product, and 34% of the anticoagulant activity was retained in vitro. The staining method for sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） was improved to obtain perfect electrophoretic pattern in less than 5min. More accurate molecular weight was deduced due to the use of PEGs as molecular weight standards.
Hirudin，凝血酵素的大多数有势力禁止者在自然发现了，在浆液有短半衰期，它显著地作为抗凝剂限制它的临床的申请。最近， PEGylation 通常被用作一个有效方法在浆液延长它的半衰期。与在随机指向离氨酸残余的基本条件下面的非特定的 PEGylation 相对照，在略微酸的条件下面的 PEGylation 地点最好指向 histidine 残余，并且仅仅在 r-hirudin 在 51 点有一 histidine 残余；因此， succinimidyl 羰基 methoxy 聚乙烯乙二醇(SC-mPEG， 20000 ) 在略微酸的 pH 被纳入 r-hirudin 赞成 mono-PEGylated r-hirudin 的形成。有高 mono-PEGylated 比率的反应混合被一步舞离子交换容易分开色析法(IEC ) 过程。约 79.71% mono-PEGylated r-hirudin 是在 His51 的 PEGylated，它证明酸的 PEGylation 操作原则上阻止了 r-hirudin 的活跃中心(Lys47 ) 的 PEGylation。有比 95% 高的纯净的 Mono-PEGylated 产品作为占优势的产品被获得，并且 34% 抗凝剂活动在 vitro 被保留。为钠 dodecyl 硫酸盐 polyacrylamide 胶化电气泳动(SDS 页) 的染色的方法被改进在不到 5min 获得完美的 electrophoretic 模式。更精确的分子的重量作为分子的重量标准由于木钉的使用被推出。
