Molecular interaction of bovine kininogen and its derivatives with papain

The molecular interaction of bovine kininogen and its derivatives with papain was investigated. High-molecular-weight kininogen (HMWK) or low-molecular-weight kininogen (LMWK) and inactive papain treated with N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl] agmatine (E-64) formed, respectively, a complex, which was dissocia ble on sodium dodecyl sulfate polyacrylamide-gel electrophoresis (SDS-PAGE). The densitometric determination of the bands separated on SDS-PAGE and amino acid analysis of the samples extracted from the electrophoresis gel revealed that the complex between kininogen and papain is formed in a molar ratio of one to one. Moreover, analysis of the inhibition of the caseinolytic activity of papain by these kininogens indicated that HMWK, LMWK, and kinin-free derivatives obtained from both kininogens inhibit active papain with a stoichiometry of 1 1. On the other hand, the papain activity was inhibited by two kinds of cyanogen bromide fragments isolated from the heavy chain of HMWK. These two fragments with K₁ values of 38 and 0.64 nM corresponded, respectively, to residue Nos. 47 to 243 and Nos. 244-360 of the HMWK heavy chain. These results suggest that in the intact HMWK and LMWK, one of the two potential reactive sites interacts with papain to form a complex and that the other reactive site becomes active only after separation of the two sites.