STUDIES ON PORCINE HIGH-MOLECULAR-WEIGHT KININOGEN - AN IMPROVED METHOD FOR THE PURIFICATION OF PORCINE HIGH-MOLECULAR-WEIGHT KININOGEN ANDCLEAVAGE OF THE KININOGEN BY THE ACTION OF PORCINE PLASMA KALLIKREIN

By introduction of stepwise DEAF Sephadex A-50 and copper-Chelating Se pharose 6B column chromatographies, about 18.5 mg of high molecular we ight kininogen (HK) composed of a single polypeptide chain was obtaine d from 500 ml of porcine plasma. Molecular weights of reduced or non-r educed preparation were estimated to be 110 kDa and 116 kDa, respectiv ely, by SDS-PAGE. Using the preparation, cleavage of HK by porcine pla sma kallikrein (KK) was investigated. A single polypeptide HK was clea ved into two chains cross-linked by disulfide bond(s), accompanying th e release of kinin. Further degradation was not observed. Molecular we ights of heavy-chain (H-chain) and light-chain (L-chain) were estimate d to be 61 kDa and 56 kDa, respectively, by SDS-PAGE. The amino- (N-) terminal sequences of intact HK, reduced and carboxymethylated- (RCM-) H-chain, RCM-L-chain and the peptide around the kinin moiety obtained by BrCN digestion were determined. Their sequences were highly homolo gous with those of bovine or human HK. These results indicate that pla sma KK first cleaved the Arg-Ser bond of HK, and formed nicked HK. The second cleavage yielded bradykinin (BK) and kinin-free protein, which was apparently of equal size to the nicked HK. The structure of HK wa s from the N-terminus to the carboxy- (C-) terminus, H-chain-BK-L-chai n, (C) 1998 Elsevier Science Inc. All rights reserved.