Purification and Characterization of Kininogens from Sheep Plasma
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High molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK) have been purified from sheep ( Avis Arias) plasma in three steps involving ammonium sulphate precipitation, column chromatography on Sephacryl-300HR and ion exchange chromatography on DEAE cellulose. HMWK gave a single band on native and SDS-PAGE with a molecular weight corresponding to 280 kDa. Under reducing conditions purified HMWK was again resolved to a single band with molecular weight corresponding to 140 kDa indicative of its dimeric nature. LMWK was resolved into two isoforms named as LMWK1 and LMWK2, with an apparent molecular weight of 68 kDa. The yield of HMWK, LMWK1 and 2 was about 8.1, 5.63 and 10.65 respectively. HMWK, LMWK1 and 2 strongly inhibited activities of ficin and papain but not of trypsin, chymotrypsin and bromelain. Ki values estimated for HMWK with papain and ficin was 0.8 and 0.6 nM respectively. Ki values estimated for LMWK1 and 2 with papain were 2.40 and 2.00 nM respectively. Binding of HMWK, LMWK1 and 2 to activated papain were accompanied by pronounced changes in secondary and tertiary structure that are compatible with perturbations of environment of aromatic residues.
KeywordsAvis Arais cysteine proteinase characteristics kininogen purification
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