Large scale purification of isoinhibitors of trypsin from swine colostrum using zinc chelate chromatography and chromatofocusing.
Low molecular weight trypsin inhibitors were purified from swine colostrum on a large scale under mild conditions. Ammonium sulfate fractionation and metal chelate chromatography of zinc chelate Sepharose and phenyl Sepharose were used for removal of the bulk of proteins. The inhibitors showed only a weak hydrophobic interaction with phenyl Sepharose even in the presence of 1 M (NH4)2S04, and advantage was taken of this property to remove the inhibitors from contaminating colostrum proteins which remained tightly adsorbed to phenyl Sepharose under these conditions. The low and high molecular weight inhibitors were then separated by gel filtration on Bio-Gel P-300. The low molecular weight material was eluted in three major inhibitor fractions on DEAE-Sepharose. Chromatofocusing of these fractions provided greater resolution of the inhibitors, and several previously unreported inhibitor peaks were detected. The six major inhibitors purified by chromatofocusing were homogenous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. These inhibitors were composed of a single polypeptide chain with a molecular weight of 18,000 as determined by Sephacryl S-200 gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and beta-mercaptoethanol. The specific activities of the pure inhibitors were approximately 30% higher than those previously reported.
D000818:Animals; D002850:Chromatography, Gel; D003126:Colostrum / Q000032:analysis*; D005260:Female; D007202:Indicators and Reagents; D011247:Pregnancy; D012685:Sepharose / Q000031:analogs & derivatives; D013552:Swine; D014361:Trypsin Inhibitors / Q000302:isolation & purification*
D007202:Indicators and Reagents; D014361:Trypsin Inhibitors; C030523:zinc chelate-sepharose; D012685:Sepharose