Characterization of guinea-pig high-molecular-weight kininogen as multi-functional molecule.
High-molecular-weight (HMW) kininogen was purified from guinea-pig plasma by measuring its ability to correct the prolonged clotting time in human HMW kininogen deficient plasma (Fitzgerald trait). The purified HMW kininogen demonstrated a homogeneous band in disc gel electrophoresis in the presence of sodium dodecyl sulfate under reducing or non-reducing conditions with an apparent molecular weight of 100,000. Kinin released from HMW kininogen by treatment with guinea-pig plasma kallikrein was identified as bradykinin by reverse-phase HPLC and amino-acid analysis. The capacity of HMW kininogen as a thiol-proteinase inhibitor was realized by its dose-dependent inhibitory activity to papain. The Ki value for papain was estimated to be 42 pM. The kinin-free HMW kininogen maintained the inhibitor and clotting-factor activities with similar capacities to those of the HMW kininogen molecule. Heavy chain (H-chain) and light chain (L-chain) of HMW kininogen were prepared from reduced and alkylated kinin-free HMW kininogen by HPLC. The S-alkylated H-chain, but not L-chain, demonstrated the inhibitor activity with the Ki value 6.9 nM for papain, whereas the S-alkylated L-chain, but not H-chain, maintained the clotting activity one-third of the capacity of HMW kininogen. Specific antibodies recognized HMW kininogen, but also a probable low-molecular-weight kininogen(s) with an apparent molecular weight of 60,000 in the guinea-pig plasma. All of these properties are consistent with the reports on human, bovine and rat HMW kininogen.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001777:Blood Coagulation; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006168:Guinea Pigs; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D007700:Kinetics; D007704:Kininogens / Q000097:blood* / Q000302:isolation & purification / Q000502:physiology; D007705:Kinins / Q000032:analysis; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D010206:Papain / Q000037:antagonists & inhibitors
D000596:Amino Acids; D007704:Kininogens; D007705:Kinins; D046911:Macromolecular Substances; D010206:Papain