Isolation and characterization of hirudin isoinhibitors and sequence analysis of hirudin PA.

  title={Isolation and characterization of hirudin isoinhibitors and sequence analysis of hirudin PA.},
  author={Johannes Dodt and Werner Machleidt and Ursula Seem{\"u}ller and Reinhard Maschler and Hans Fritz},
  journal={Biological chemistry Hoppe-Seyler},
  volume={367 8},
A five-step isolation procedure has been developed for the purification of isoforms of hirudin (isohirudins) from whole leeches. The final purification of two thrombin-inhibiting preparations by reversed-phase high-performance liquid chromatography yielded several isohirudins with either N-terminal valine or isoleucine but with identical inhibition characteristics, i.e. specific thrombin inhibiting activities of 680-720 IU/mg and dissociation constants Ki of the thrombin-inhibitor complexes… 
C-terminal proteolytic degradation of recombinant desulfato-hirudin and its mutants in the yeast Saccharomyces cerevisiae.
It was concluded that ysc alpha might also be responsible for the C-terminal degradation of recombinant atrial natriuretic factor and epidermal growth factor expressed in yeast.
Use of fragments of hirudin to investigate thrombin-hirudin interaction.
Hirudin and the N-terminal fragment completely prevented the cleavage of alpha-thrombin by pancreatic elastase while the C- terminal fragment afforded a lesser degree of protection.
Molecular basis for the inhibition of thrombin by hirudin.
The amino acid sequence of the 65 residue major form of hirudin, originally isolated from the salivary glands of the European medicinal leech Hirudo medicinalis, is shown and one of the charged residues found in this region of the natural hirUDin is a sulphated tyrosine residue at position 63.
Identification and characterization of hirudin-HN, a new thrombin inhibitor, from the salivary glands of Hirudo nipponia
The N-terminal structure of the mature hirudin-HN protein was shown to be important for anticoagulant activity by comparing the activity and thrombin affinity of Hir and M-Hir.
Contribution of the N-terminal region of hirudin to its interaction with thrombin.
It appears that a positive charge immediately adjacent to the N-terminal hydrophobic residue is required for optimal binding to thrombin.
Purification and biochemical characterization of recombinant hirudin produced by Saccharomyces cerevisiae.
UV spectral analysis suggested that, in contrast to the natural molecule, recombinant hirudin produced by S. cerevisiae is not sulfated.
Hirudin and hirudin-based peptides.
Influence of chain shortening on the inhibitor properties of hirudin and eglin c.
To find out minimal sizes of the proteinase inhibitor proteins hirudin and eglin necessary for their biological activity the inhibitors were incubated with exopeptidases and complex of thrombin with hirUDin lacking 3 C-terminal amino-acid residues showed a 15-20-fold increased Ki value.
Preparation of monoclonal antibodies to hirudin and hirudin peptides
A panel of four monoclonal antibodies was obtained against hirudin, a potent and specific inhibitor of thrombin, by immunizing three groups of mice with protein conjugates made of recombinant


Cloning and expression of a cDNA coding for the anticoagulant hirudin from the bloodsucking leech, Hirudo medicinalis.
The hirudin cDNA was expressed in Escherichia coli under the control of the bacteriophage lambda PL promoter and the recombinant product is biologically active, inhibiting the cleavage by thrombin of fibrinogen and a synthetic tripeptide substrate.
The complete covalent structure of hirudin. Localization of the disulfide bonds.
Hirudin seems to be unique in its primary structure and hence designates an unknown inhibitor family, due to the lack of any closer sequence homology and topological structural homology to other serine proteinase inhibitor proteins.