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| Hirudin is the most potent inhibitor of thrombin found in nature. As a promising anticoagulant, a significant drawback which significantly inhibits its clinical application, is the short serum half-life. In order to prolong circulation half-life in vivo, PEGylation is an effective and commonly used method. In this study Succinimidyl carbonyl mPEG (SC-mPEG) 20kDa was attached to recombinant hirudin (r-Hir) at mildly acidic pH to favor the formation of mono- PEGylated r-Hir, since histidine residues represent the preferred PEGylation sites under mildly acidic conditions, and there is only one histidine residue, His51, in r-Hir. Because of the high ratio of monopegylated product, the reaction mixture was easily separated by a one-step ion-exchange chromatographic(IEC) separation procedure. The proportion of urethane bonds involving carboxyalkylated histidines was assayed using its sensitivity to neutral hydroxylamine, and about 79.71% of the mono-substituted PEGylated r-Hir was PEGylated |
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| Keywords:r-Hir; histidine residues; SC-mPEG; monopegylation; pH; anticoagulant activity |
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