Characterization, stability and refolding of recombinant hirudin.


A recombinant variant of hirudin, the blood-clotting inhibitor of the leech Hirudo medicinalis, has been characterized employing spectroscopic and hydrodynamic techniques. Conditions have been defined for efficient reconstitution of the native, disulfide-bonded inhibitor from completely unfolded, reduced polypeptide chains. The spectral properties of the native inhibitor are consistent with previous results on the solution structure of hirudin. Extremely low circular dichroism in the far ultraviolet ([theta]Mr,220 nm = -8 +/- 1 x 10(2) deg.cm2.dmol-1) indicates a very low content of regular secondary structure. Although both tyrosine residues of the recombinant inhibitor titrate around pH 10.6, typical for solvent-exposed tyrosines, fluorescence emission and near-ultraviolet circular dichroism suggest that at least one of the tyrosines is partially shielded from solvent quench, and immobilized in an asymmetric environment. Reversible thermal unfolding of hirudin around 65 degrees C is indicated by the disappearance of its dichroic absorption in the near ultraviolet and by a fourfold increase in ellipticity at 225 nm. The transition can be approximated by a two-state model with a transition enthalpy of delta Hvan't Hoff = 159 kJ/mol and a transition entropy of 464 J.mol-1.K-1. Reduced hirudin at room temperature is largely unfolded and inactive as an inhibitor of thrombin assayed with a low-molecular-mass substrate. Refolding and reoxidation are observed at alkaline pH in the presence of a mixture of glutathione and glutathione disulfide. Spectroscopy, thrombin inhibition, and reversed-phase HPLC indicate reconstitution yields close to 100% and that the reconstituted inhibitor is identical to the native starting material. Study holds ProTherm entries: 7171, 7172 Extra Details: disulfide-bonded inhibitor; secondary structure;,solvent-exposed tyrosine; reversed-phase HPLC

Submission Details


Submitter: Connie Wang

Submission Date: April 24, 2018, 8:33 p.m.

Version: 1

Publication Details
Otto A;Seckler R,Eur. J. Biochem. (1991) Characterization, stability and refolding of recombinant hirudin. PMID:1935981
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Hirudin variant-1 P01050 HIRV1_HIRME
96.9 Hirudin variant-1 P28511 HIR3C_HIRME
96.9 Hirudin variant-1 P28509 HIR3_HIRME
93.8 Hirudin variant-1 P28508 HIR3P_HIRME
95.4 Hirudin variant-1 P28510 HIR3B_HIRME
92.3 Hirudin variant-1 P28506 HIR2B_HIRME
92.3 Hirudin variant-1 P28503 HIR2A_HIRME
90.8 Hirudin variant-1 P28504 HIR2_HIRME
98.0 Hirudin variant-1 P84590 HIRUD_POEVI