title: State of aggregation of recombinant hirudin in solution under physiological conditions.
abstract: The state of aggregation of recombinant desulfatohirudin (r-HV1) in solution under physiological conditions (pH 7.5, 0.15 N NaCl) was investigated by sedimentation equilibrium. The weight-average molecular weight MW determined by sedimentation equilibrium was found to be 6914 +/- 76 Da compared to 6964 Da expected from the amino acid sequence. The MZ/MW ratio was found to be 1.03, which demonstrates that under the conditions studied hirudin exists in solution as a monomer. This result is in agreement with the relative molecular weight (M,) of recombinant hirudin variant 3 reported by Otto and Seckler [(1991), Eur. J. Biochem. 202, 67-73], who also used equilibrium ultracentrifugation, but not with the molecular weight estimated from gel permeation chromatography of natural hirudin (51,300 Da) [Konno et al. (1988), Arch. Biochem. Biophys. 267, 158-166]. Knowledge of the state of aggregation is essential for understanding the mechanism of interaction of thrombin and hirudin under physiological conditions.
authors.affiliation: Cornell Center of Advanced Technology in Biotechnology, Cornell University, Ithaca, New York 14853-2703, USA.
authors.forename: T W
authors.initials: TW
authors.lastname: Thannhauser
authors.forename: H A
authors.initials: HA
authors.lastname: Scheraga
mesh_terms: D000818:Animals; D002850:Chromatography, Gel; D006629:Hirudins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007865:Leeches / Q000378:metabolism; D008970:Molecular Weight; D009579:Nitrobenzoates / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013431:Sulfates / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D014461:Ultracentrifugation
chemical_list: D006629:Hirudins; D009579:Nitrobenzoates; D011994:Recombinant Proteins; D013431:Sulfates; D014443:Tyrosine; C041724:2-nitro-5-thiosulfobenzoic acid