This chapter presents a procedure for purification of plasma prekallikrein and its assay technique. Purification is difficult by conventional methods of protein fractionation because its concentration in plasma is low and it is very sensitive to protease digestion. In the isolation procedure described in this chapter, benzamidine-agarose and agmatine- or arginine methyl ester-agarose column chromatography play an important role. Purification of human plasma prekallikrein involve: ammonium sulfate fractionation and chromatography on diethylaminoethyl (DEAE)sephadex of fresh-frozen human plasma; subsequent chromatography on the second DEAEsephadex, heparin-agarose, carboxymethyl -sephadex, concanavalin A-agarose, and agmatine-agarose. Each purification step is performed in the presence of protease inhibitors to restrict activation of the precursor form. For the coagulant assay of plasma prekallikrein, activity is calculated from a calibration curve where the log ofprekallikrein concentration is plotted against the log of the clotting time. In amidase assay kallikrein is measured by using the chromogenic kallikrein substrate Bz-Pro-Phe-Arg-p-nitroanilide after activation of prekallikrein by trypsin.
ASJC Scopus subject areas
- Molecular Biology