Purification of human low molecular weight kininogen and its application for a simple ND sensitive method for determination of human urinary kallikrein activity.
Human plasma low molecular weight kininogen was purified with ammonium sulfate fractionation, DEAE-cellulose, CM-Sephadex C-50 and aprotinin-agarose affinity column chromatography, after which this was further purified with Sephadex G-150 and DEAE-Sephadex A-50 column chromatographies. Kallikrein activity was measured as the kininogenase activity reflecting the kinin-producing capacity from kininogen. The purification factor from crude plasma to purified substrate was 44-fold, and the recovery was 18%. The purified human substrate did not contain kinin-generating or destroying enzymes which would interfere with kininogenase activity, and showed a cross-reactivity of less than 0.1% against kinin antiserum. In the kininogenase assay, all kininogen was removed by adding ethanol to terminate the enzyme reaction. Because of the high sensitivity of kinin radioimmunoassay, the kinin levels in urine could be determined in very small amounts of samples (0.5 to 2.0 nl of the original urine). These findings indicated that kinin levels in incubation solution could be measured directly, and the control tubes are unnecessary in this assay procedure. In a comparison among human, dog and bovine low molecular weight kininogen as the substrate for human urinary kallikrein, the enzyme activity was 5 and 80 fold higher in the human low molecular weight kininogen, respectively, suggesting that a human substrate is the best for human enzymes. This simple, specific, sensitive and homologous kininogenase assay system seems to be very useful investigating the physiological or pathophysiological role of the renal kallikrein-kinin system in hypertensive and renal diseases.
D000818:Animals; D002852:Chromatography, Ion Exchange / Q000379:methods; D004285:Dogs; D006801:Humans; D006929:Hyperaldosteronism / Q000201:enzymology; D007610:Kallikreins / Q000378:metabolism / Q000652:urine*; D007700:Kinetics; D007704:Kininogens / Q000097:blood* / Q000302:isolation & purification; D008970:Molecular Weight; D012016:Reference Values
D007704:Kininogens; D007610:Kallikreins