Isolation of plasma kallikrein by high efficiency affinity chromatography and its characterization

We established a simple method for the purification of human plasma kallikrein (PK) by affinity chromatography and characterized it by analytical reverse phase-HPLC and Time of Flight Mass Spectroscopy (TOF-MS). The affinity resin (PKSI-Toyopearl) was synthesized using a selective synthetic inhibitor of plasma kallikrein (PKSI-527) as a ligand. The resin was found to have the highest efficiency in PK purification when the coupling ratio of PKSI-527 per resin was 9-14 micromol/g. PK was purified 466-fold with a yield of 83% from acetone-activated human plasma by affinity chromatography. The purity of PK thus obtained was confirmed by reverse phase-HPLC with a linear gradient of acetonitrile. The molecular weight of the purified PK was determined to be 86,151 by TOF-MS.