A convenient large-scale preparation of high molecular weight kininogen from human plasma.

Abstract

Human high molecular weight (HMW) kininogen was purified by chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50, followed by gel filtration on Sephadex G-50. From 5 l fresh human plasma approximately 120 mg HMW kininogen was obtained. The yield was 40%. The preparation had a specific activity of 14 microgram bradykinin equivalent/A280 unit. Upon polyacrylamide disc gel electrophoresis HMW kininogen was separated into two close bands, whereas only one band with an apparent Mr of 120 000 was obtained in sodium dodecyl sulfate electrophoresis. Both protein fractions separated in disc gel electrophoresis released kinins upon incubation with kallikreins. The purified HMW kininogen had an isoelectric point of 4.65 when measured by isoelectric focusing. The amino acid composition of the purified HMW kininogen is given. The amino terminus of the molecule is blocked. In oligomerization studies adducts with molecular weights up to 810 000 were obtained. HMW kininogen gave a single precipitin arc in immunoelectrophoresis with antiserum directed against HMW kininogen.