Immobilization of the thrombin inhibitor r-hirudin conserving its biological activity.

Surface immobilization of the thrombin inhibitor r-hirudin was carried out on two different polymers. Linkage to poly(urethane-graft-acrylic acid) (PAC/PU) was done via carboxylic acid groups, using a water soluble carbodimide, while the immobilization on a modified poly[(ethene-co-vinyl acetate)-graft-vinyl chloride] (PVC/EVA) was achieved via the alcohol groups of the polymer using HDI as spacer. Direct immobilization of r-hirudin leaded to a remarkable loss of thrombin activity. As proved by means of protein chemical analysis, loss of activity was due to a selective coupling via the N-terminal amino group of r-hirudin, which is essential for its thrombin activity. Based on these results we developed an immobilization method via an epsilon-amino group of r-hirudin preserving full biological activity of the r-hirudin coated surface. Copyright 2001 Kluwer Academic Publishers