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.
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Federal Republic of Germany.
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Otto
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Seckler
D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000378:metabolism; D004355:Drug Stability; D005978:Glutathione / Q000494:pharmacology; D006629:Hirudins / Q000737:chemistry* / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000037:antagonists & inhibitors; D014461:Ultracentrifugation
D004220:Disulfides; D006629:Hirudins; D011994:Recombinant Proteins; D013917:Thrombin; D005978:Glutathione