Characterization, stability and refolding of recombinant hirudin

Abstract

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 nm = -8 f 1 x lo2 deg . cm2 . dmol-') 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 "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 AHvan3, Hoff = 159 kJ/mol and a transition entropy of 464 J . mol-' . K-' . 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.

Restoring the Procofactor State of Factor Va-like Variants by Complementation with B-domain Peptides

“…4B). In the absence of FV-810, the sedimentation coefficient (s 20,w 0 ) of QSY7-BR was 0.98 s, identical to the previously determined value for recombinant hirudin, which is similar in size (34). In the presence of FV-810, the QSY7-BR sedimentation coefficient shifted to 9.8 s, somewhat larger than our previously determined value for FV-810 of 8.43 s (15).…”

State of aggregation of recombinant hirudin in solution under physiological conditions

“…This result for r-HV1 may be assumed to apply also to the natural material even though the recombinant protein is not sulfated at Tyr64. Similar conclusions were reported by Otto and Seckler (1991) for a recombinant variant that was 82% homologous to r-HV1. Presumably, the high value obtained from the chromatographic measurements is due to errors that can arise in this technique because it does not account adequately for protein shape or various types of interaction between the protein and the chromatographic medium, e.g., hydrophobic or hydrophilic affinity, ion exchange or ion exclusion effects, etc.…”

“…Both forms are highly inhibitory, the natural form being a slightly better inhibitor of thrombin than the recombinant form, K~ = 2 • 10 -14 and 2 x 10 -~3 M, respectively (Braun et aL, 1988). Recently, recombinant hirudin (residues 1-65) and the amino-terminal fragment of hirudin (residues 1-49) have been used as model systems with which to study the process of protein folding (Otto and Seckler, 1991;Chang, 1992, 1993;Chang, 1993Chang, , 1994Thannhauser and Scheraga, 1997). It has been reported that HV1 exists in solution as a multimeric aggregate with a molecular weight of 51,300 Da (Konno et al, 1988).…”

Chemical synthesis and structural characterization of the RGD‐protein decorsin: A potent inhibitor of platelet aggregation

“…Nevertheless, the far-UV CD spectrum of decorsin does not show a negative band at 215-220 nm and a positive one at 195-200 nm typical of a /?-protein (Brahms & Brahms, 1980;Johnson, 1990) and is different from that reported for intact hirudin HV1 (Otto & Seckler, 1991) or for the NH2-terminal domain 1-47 of hirudin HM2 (De Filippis et al, 1995). Because the CD signal due to the peptide chromophore in the far-UV region can be masked by the contributions of aromatic residues and disulfide bonds (Manning &Woody, 1989;Vuilleumier et al, 1993), we recorded the far-UV CD spectra of model compounds acetyl-tyrosinamide (Ac-Tyr-NH2), acetyl-phenylalanylamide (Ac-Phe-NH2) and cystine (Fig.…”