PURIFICATION AND HETEROGENEITY OF HUMAN KININOGEN.

Wiley - Tập 7 Số 3 - Trang 261-280 - 1975
Ulla Hamberg1,2, Peter Elg1, Erkki Nissinen1, Patrick Stelwagen1
1Department of Biochemistry, University of Helsinki, Helsinki, Finland
2Part of these results were reported in the Sympsoium on “Biological and Pharmacological Properties of Peptides” (Polish Academy of Sciences), Bialowieza, Poland, September 8–9, 1972, and in the International Conference on “Chemistry and Biology of the Kallikrein-Kinin System in Health and Disease” (The John E. Fogarty International Centre and National Heart and Lung Institute), Reston, Virginia, USA, October 20–23, 1974.

Tóm tắt

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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Wiley

Tập 7 Số 3

261-280

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