The trypsin binding was compared with preparations of a,-macroglobulin obtained from human plasma, serum or Cohn's fraction I11 by ammonium sulphate precipitation. Determination of the binding ratio a t various purification steps showed close to 1 : l molecular proportion, regardless of the degree of purity of the preparations. The a,-macroglobulin * trypsin complex was separated by gel chromatography on Sephadex G-200, using standardized conditions and estimating the bound and free trypsin in the eluate fractions. It was found that activated plasmin interfered with trypsin binding of a,-macroglobulin from Cohn's fraction 111.Immunologically pure a,-macroglobulin was obtained in six preparative steps from whole plasma after applying zone electrophoresis in polyvinylchloride. The protein was characterized by sedimentation rate, immunochemical analyses, isoelectric focusing, amino acid analysis and trypsin binding.Decreased binding capacity towards trypsin and plasmin could be detected after lyophylization of a,-macroglobulin. Comparison with low-temperature denaturation in sedimentation studies suggests that the functionally intact protein was not restored in spite of goo/, reaggregation of the 11-12-5 subunit. The structure dependence of the enzyme binding is discussed.The a,-macroglobulin is one of the major inhibitors of proteolytic activity in human plasma. The protein was isolated and characterized by Schultze et aZ. The earlier studies on complex formation with trypsin and some blood coagulation enzymes were done with partially purified a,-macroglobulin, serum or whole plasma. Recent purifications have aimed mainly a t preparing the native, immunologically pure protein . Such discrepancies and the fact that the trypsin or plasmin binding activity has not previously been studied in parallel with the purification of the human a,-macroglobulin [ 181 show that characterization of the functionally intact, purified protein is still incomplete. This report, dealing with the comparison of various methods to purify a,-macroglobulin provides information concerning the binding activity a t various stages of purification. Human whole plasma, serum or Cohn's fraction I11 precipitated with ammonium sulphate were used as starting materials. The amidase activity of the complex with trypsin was measured on N-benzoyl-DL-arginine p-nitroanilide [19,20], esterase activity on p-toluenesulphonyl-L-arginine methyl ester [8,9]. I n each case the complex was first separated by gel chromatography on Sephadex G-200. A decrease of the molecular binding activity was found with denatured a,-macroglobulin. The purified protein was characterized by sedimentation rate, immunological analyses and isoEur.
Various methods of preparing human kininogen were investigated with an aim to limit the immunoreactive contaminant proteins to permit purification by immunosorption. A five‐step procedure is described giving 7.5% yield of highly purified kininogen (pharmacological purity 14–20) from pooled human plasma, and containing approximately 30% alpha‐2HS‐glycoprotein and 2.8% albumin. Alpha‐2HS could not be removed by polyacrylamide gel electrophoresis or isoelectric focusing in column. Analysis of heterogeneity of kininogen after chromatography on DEAE‐Sephadex using various linear gradients and gel filtration on Sephadex G‐100 suggested that a minor component may be an aggregate, not included in the yield. It remains uncertain whether this component derives from an occasionally observed high molecular form of active kininogen in the primary purification steps in the 7–12 S sieve fractions from Sephadex G‐200, and excluded from further purification by pooling. Purification with immunosorbents was investigated using batch operations with antibody specific polymers prepared with antisera insolubilized with ethylchloroformate. It was found that the adsorption‐desorption procedure was favourable for immunization purposes in producing highly specific immunologically pure kininogen. The kininogen obtained by this method or by the removal of contaminant alpha‐2HS and albumin with the corresponding antibody specific polymers gave similar heterogenous patterns by polyacrylamide gel electrophoresis, indicating a main band of kininogen and several faintly stained bands which responded only to anti‐kininogen. With 200 μg of the kininogen protein purified by immunosorption using monospecific antiserum the kininogen precipitation titre was 1:8 after 6–8 weeks in rabbits. With a polymer prepared with 4 ml anti‐kininogen serum (1:8) and incubated with 800 μg highly purified kininogen approximately half the protein was desorbed with 2 M and 3 M sodium iodide in the first adsorption‐desorption procedure.
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