Hans-Gregor Gattner

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 ε-amino group of r-hirudin preserving full biological activity of the r-hirudin coated surface.

[A14-Phenylalanine]Insulin: A New Synthetic Analogue

An analogue of porcine insulin which differs from the native molecule in that the tyrosine residue in Pos. A19 is replaced by phenylalanine has been synthesized. The [PheA19]A chain was synthesized by the fragment condensation and purified as tetra-S-sulfonate by ion-exchange chromatography on DEAE-cellulose at pH 3.5. Conversion of the latter into the sulfhydryl form and combination with native sulfhydryl B chain yielded the [PheA19]insulin, which was purified by ion-exchange chromatography on CM-cellulose at pH 4.0 with an linear NaCl gradient. The biological activity of this analogue was 22.6% as measured by the rat epididymal adipocytes. It was suggested that in the insulin molecule the hydroxyl function of A19-tyrosine participates in an hydrogen-bond with the carbonyl function of A1-glycine. That hydrogen bond formation is critical for the stability of the hormone-receptor complex. The low biological activity found by us supports this hypothesis. The circular dichroism data in far UV suggest that the introduction of phenylalanine leaves the molecule structure essentially undisturbed, however the change observed in near UV could be accounted for the exchange.