Protein-protein interactions in contact activation of blood coagulation. Characterization of fluorescein-labeled human high molecular weight kininogen-light chain as a probe.
Limited proteolysis of high molecular weight kininogen by kallikrein resulted in the generation of an inactive heavy chain of Mr = 64,000 and active light chains of Mr = 64,000 and 51,000 when analyzed by sodium dodecyl sulfate (SDS)-gel electrophoresis under reducing conditions. Starting with kininogen from outdated plasma, a light chain with an apparent molecular weight of 51,000 on 7.5% SDS gels was purified and characterized. Molecular weights of 28,900 +/- 1,100 and 30,500 +/- 1,600 were obtained by gel filtration of the reduced and alkylated protein in 6 M guanidine HCl and equilibrium sedimentation under nondenaturing conditions in the air-driven ultracentrifuge, respectively. The light chain stained positively with periodic acid-Schiff reagent on SDS gels indicating that covalently attached carbohydrate may be responsible for the anomalously high molecular weight estimated by SDS-gel electrophoresis. A single light chain thiol group reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) in the presence and absence of 6 M guanidine HCl. Specific fluorescent labeling of the thiol group with 5-(iodoacetamido)fluorescein (IAF) occurred without loss of clotting activity. Addition of purified human plasma prekallikrein to the IAF-light chain resulted in a maximum increase in fluorescence anisotropy of 0.041 +/- 0.001 and no change in the fluorescence intensity. Fluorescence anisotropy measurements of the equilibrium binding of prekallikrein to the IAF-light chain yielded an average Kd of 17.3 +/- 2.5 nM and stoichiometry of 1.07 +/- 0.07 mol of prekallikrein/mol of IAF-light chain. Measurements of the interaction of prekallikrein with iodoacetamide-alkylated light chain using the IAF-light chain as a probe gave an average Kd of 16 +/- 4 nM and stoichiometry of 1.0 +/- 0.2 indicating indistinguishable affinities for prekallikrein.
D001777:Blood Coagulation / Q000187:drug effects*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005452:Fluoresceins / Q000378:metabolism; D005454:Fluorescence Polarization; D006801:Humans; D007610:Kallikreins / Q000378:metabolism*; D007704:Kininogens / Q000378:metabolism*; D008433:Mathematics; D008970:Molecular Weight; D011288:Prekallikrein / Q000378:metabolism*
D005452:Fluoresceins; D007704:Kininogens; C015296:5-iodoacetamidofluorescein; D011288:Prekallikrein; D004228:Dithionitrobenzoic Acid; D007610:Kallikreins