title: Analysis of the secondary structure of hirudin and the mechanism of its interaction with thrombin.
abstract: Highly purified hirudin with a specific activity of 13,950 antithrombin units/mg was isolated from a commercial preparation by reversed-phase chromatography. The circular dichroism (CD) spectrum of hirudin was investigated and it was found that the spectrum cannot be accounted for solely in terms of the traditional three components of peptide backbone. It was also found that the CD spectrum of the thrombin-hirudin complex was not additive with respect to the individual spectra of thrombin and hirudin. This deviation from additivity was significant between 210 and 225 nm, indicating alterations in the secondary structures of the proteins during complex formation. When thrombin was titrated with hirudin, the spectral deviation from additivity was sigmoidal, suggesting the cooperative nature of the binding process. Gel filtration of the thrombin-hirudin mixture showed no molecular species greater than a 1:1 complex (Mr 45,500), but gel filtration of free hirudin showed a multimeric form (Mr 51,300) under the same experimental conditions. It is concluded that the cooperative nature of the binding process is due to the binding of thrombin molecules to the multimeric form of hirudin. This initial binding occurs with little or no change in the CD spectrum. In the second step, the multiple complex dissociates to form 1:1 complexes, resulting in larger conformational changes and a considerable increase in binding affinity.
authors.affiliation: Department of Biochemistry, New York Medical College, Valhalla 10595.
authors.forename: S
authors.initials: S
authors.lastname: Konno
authors.forename: J W
authors.initials: JW
authors.lastname: Fenton
authors.forename: G B
authors.initials: GB
authors.lastname: Villanueva
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006629:Hirudins / Q000378:metabolism*; D006801:Humans; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism*
chemical_list: D006629:Hirudins; D013917:Thrombin