Abstract
In this chapter, a purification method of human factor XII is described that is reproducible and suitable for obtaining milligram amounts of protein. For purification frozen outdated human plasma is used, including following procedures: ammonium sulfate fractionation; first DEAE-Sephadex column chromatography; second DEAE-Sephadex column chromatography; QAE-Sephadex column chromatography; CM-celhdose column chromatography; and separation of Factor XII and Factor XIIa by Benzamidine-Agarose column chromatography. The clotting activity of factor XII is determined by a kaolin partial thromboplastin time using human factor XII-deficient plasma. The clotting time is determined by continuous tilting of the tubes at 37°C. The units of factor XII activity are calculated from a standard calibration curve prepared by a serial dilution of pooled normal human plasma. Amidase activity of factor XIIa is measured using the synthetic peptide substrate D-prolyl-L-phenylalanyl-L-arginine-p-nitroaniline.
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