Bovine Plasma KininogensI. Further Purification of High Molecular Weight Kininogen* and Its Physicochemical Properties

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Abstract
A method was developed for the further purification of high molecular weight (HMW) kininogen (previously named kininogen-I*) from bovine plasma. The method involved ion exchange chromatography on columns of DEAE-Sephadex A-50 and CM-Sephadex C-50 and gel chromatography on a column of Bio-Gel P-300, using hexadimethrine bromide to inhibit the conversion of prekallikrein to kallikrein [EC 3.4. 21.8] and diisopropylfluorophosphate to inhibit kallikrein activity. The yield was about 430 mg from 12.5 liters of fresh bovine plasma. The purified HMW kininogen gave a single band on sodium dodecylsulfate-polyacrylamide gel electrophoresis, a single precipitin line on immunoelectrophoresis and a symmetrical Schlieren pattern on ultracentrifugation. However, it gave two protein bands on polyacrylamide disc gel electrophoresis at pH 8.3. On the same electrophoresis after treatment of the kininogen with purified plasma kallikrein, these two bands were replaced by a single band with faster mobility. Thus, the results suggest that these two components of HMW kininogen must be the same molecular species. The sedimentation coefficient of purified HMW kininogen was 3.80 S and its molecular weight was estimated as about 7.6×104 by sedimentation equilibrium and the Archibald method. The molecular weight estimated by gel filtration on a column of Sepharose 4B in the presence of guanidinium hydrochloride was about 8.0×104. HMW kininogen had an isoelectric point of pH 4.5, as measured by the isoelectric focusing technique. Its polypeptide content was determined to be 76% by the biuret method and 83.5% by amino acid analysis, and there was a total of 581 amino acid residues per mole of protein. HMW kininogen contained a total of 12.6% carbohydrates, consisting of hexoses (4.57%), hexosamines (3.65%), and sialic acid (4.35%). The hexosamines were glucosamine and galactosamine in a molar ratio of 1.5 : 1. Thus, it was concluded that HMW kininogen is a typical glycoprotein in bovine plasma.

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