title: Interaction of factor XII, high-molecular-weight (HMW) kininogen and prekallikrein with sulfatide: analysis by fluorescence polarization.
abstract: The interaction of high-molecular-weight (HMW) kininogen, Factor XII and prekallikrein with sulfatide was studied by fluorescence polarization. Fluorescein-conjugated derivatives of HMW kininogen, Factor XII and prekallikrein were prepared by reacting the purified bovine factors with fluorescein isothiocyanate (FITC). The apparent dissociation constant (Kd) for the binding of FITC-labeled HMW kininogen (F-HMW kininogen) with sulfatide was calculated to be 3.2 (+/- 0.3) X 10(-8) M. This binding was partially inhibited by three kininogen derivatives, fragment 1 X 2, kinin-free protein and fragment 1 X 2-light chain, but not by kinin and fragment 1 X 2-free protein. In the presence of Factor XII, the binding of F-HMW kininogen with sulfatide was strongly inhibited, suggesting that the zymogen and the protein cofactor compete for the same or a closely related binding site on the sulfatide surface. In contrast, the binding of FITC-labeled Factor XII (F-Factor XII) with sulfatide was weakly inhibited by HMW kininogen but not by prekallikrein. The Kd value for binding of F-Factor XII with sulfatide was calculated to be 2.0 (+/- 0.3) X 10(-8) M. F-Prekallikrein did not interact with sulfatide. Moreover, the fluorescence polarization value of F-HMW kininogen decreased in the presence of prekallikrein, leveling off at a one-to-one molar ratio of prekallikrein to F-HMW kininogen. The Kd value for binding of F-HMW kininogen-light chain (F-light chain) with prekallikrein was calculated to be 3.8 (+/- 0.6) X 10(-8) M and the stoichiometry was estimated as 1 to 1.2 on a molar basis from the Scatchard plot.
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005174:Factor XII / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005454:Fluorescence Polarization; D005699:Galactosylceramides / Q000378:metabolism; D007610:Kallikreins / Q000378:metabolism*; D007704:Kininogens / Q000378:metabolism*; D008970:Molecular Weight; D011288:Prekallikrein / Q000378:metabolism*; D011485:Protein Binding; D013433:Sulfoglycosphingolipids / Q000378:metabolism*; D013499:Surface Properties; D013861:Thiocyanates
chemical_list: D005452:Fluoresceins; D005699:Galactosylceramides; D007704:Kininogens; D013433:Sulfoglycosphingolipids; D013861:Thiocyanates; D005174:Factor XII; D011288:Prekallikrein; D007610:Kallikreins; D016650:Fluorescein-5-isothiocyanate