Purification of prekallikrein from porcine plasma and its conversion to active kallikrein.
Plasma prekallikrein, a precursor protein of kallikrein [EC 3.4.21.8], was highly purified from porcine plasma by chromatography on a DEAE-Sephadex A-50 column, followed by rechromatography on a DEAE-Sephadex A-50 column, chromatography on a CM-Sephadex C-50 column and affinity chromatography on a p-aminobenzamidine-epsilon-aminocaproic acid-Sepharose 4B column. By this procedure, 3.3 mg of purified material was obtained from 1.6 liters of porcine plasma and about 240-fold purification was achieved from the first DEAE-Sephadex A-50 chromatography. The purified protein was found to give a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gel disc electrophoresis. This preparation did not contain kallikrein, Factor XII (Hageman factor) of the blood coagulation system, high molecular weight (HMW) kininogen or plasma kininase. Thus, the material is presumed to be functionally pure. The molecular weight of prekallikrein was estimated to be about 88,000 by SDS-polyacrylamide gel electrophoresis, and prekallikrein consists of a single polypeptide chain. Activation of prekallikrein by trypsin [EC 3.4.21.4] was found to involve the cleavage of a single peptide bond on the disulfide-bridged polypeptide chain, and no change of molecular weight was observed during the activation. This trypsin-activated kallikrein released kinin rapidly from bovine HMW kininogen. However, liberation of kinin was extremely slow from bovine low molecular weight (LMW) kininogen. The kallikrein activity was inhibited by soybean trypsin inhibitor (SBTI) and Trasylol, but not by Polybrene or egg-white trypsin inhibitor (EWTI).
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D007610:Kallikreins / Q000096:biosynthesis* / Q000097:blood*; D008970:Molecular Weight; D011288:Prekallikrein / Q000097:blood* / Q000302:isolation & purification; D013552:Swine
D011288:Prekallikrein; D007610:Kallikreins