Human high molecular weight kininogen as a thiol proteinase inhibitor: presence of the entire inhibition capacity in the native form of heavy chain

Abstract

High molecular weight (HMW) kininogen was purified from fresh human plasma by two successive column chromatographies on DEAE-Sephadex A-50 and Zn-chelate Sepharose 4B. The purified HMW kininogen appeared to be a single band on sodium dodecyl sulfate (SDS)-polyacrylamide disc gel electrophoresis in both the presence and absence of .beta.-mercaptoethanol. However, it gave two bands on nonreduced SDS-polyacrylamide slab gel electrophoresis, a major band of dimeric form (Mr 200,000, ca. 95%) and a minor band of monomeric form (Mr 105,000, ca. 5%). Under reduced conditions, the dimeric form was converted stoichiometrically to a monomeric form (Mr 110,000), and the monomeric form observed under nonreduced conditions (Mr 105000) was converted to a heavy chain (Mr 60,000) and a light chain (Mr 50,000). The formation of a dimer of HMW kininogen was also confirmed by an immunoblotting experiment. This unique property of intact HMW kininogen to form a dimer was further utilized in studies on the kininogens and derivatives as thiol proteinase inhibitors. The purified HMW kininogen strongly inhibited the caseinolytic activities of calpain I, calpain II, and papain but not those of trypsin, chymotrypsin, and thermolysin, indicating that it was a group-specific inhibitor for thiol proteinases. When HMW kininogen was reduced with 0.14 or 1.4 M .beta.-mercaptoethanol, its inhibitory activity was partially or mostly inactivated, but on subsequent air oxidation its activity was almost completely recovered. In addition, kinin-free and fragment 1,2 free HMW kininogen showed higher inhibitory activity than the intact HMW kininogen. The heavy chain exhibited the highest activity and bound with 2 mol of papain per mole of the heavy chain. These results and the sequence data previously reported on low molecular weight (LMW) kininogen [Ohkubo, I., Kurachi. K., Takasawa, T., Shiokawa, H., and Sasaki, M. (1984) Biochemistry 23, 5691-5697] clearly demonstrated that two reactive sites for thiol proteinase inhibitor activity are located in the heavy chain of HMW and LMW kininogens and that the light chain and fragment 1,2 moieties of the kininogens interfere with the inhibitory activity of the heavy chain by steric hindrance.