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.
Tada
M
M
Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Japan.
Tsuda
Y
Y
Wanaka
K
K
Hijikata-Okunomiya
A
A
Horie
N
N
Okamoto
U
U
Okamoto
S
S
Okada
Y
Y
D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007610:Kallikreins / Q000032:analysis / Q000097:blood / Q000302:isolation & purification*; D013058:Mass Spectrometry; D008970:Molecular Weight
D007610:Kallikreins