A method for the isolation and purification of physiologically active substances with the use of enzyme - enzyme inhibitor systems

摘要

1442796 Isolating and purifying proteolytic enzymes and inhibitors ONO PHARMACEUTICAL CO Ltd 18 Dec 1973 [19 Dec 1972] 58687/73 Heading C3H A method of isolating and purifying a physiologically active substance, defined as meaning either proteolytic enzymes which take part in the production-consumption of kinin or an inhibitor which is proteinaceous in nature and inhibits the activity of the above proteolytic enzyme, contained in mammalian tissue or body fluid comprises the steps of passing an extract of said tissue or a diluted solution of said body fluid through a combination of columns packed with an insolubilized naturally occurring proteolytic enzyme and an insolubilized naturally occurring proteolytic enzyme inhibitor corresponding to the enzyme, respectively, in a continuous manner to adsorb the corresponding enzyme inhibitors present in the extract or diluted solution on to the insolubilized enzyme and the corresponding enzymes present in the extract or diluted solution on to the insolubilized enzyme inhibitor, respectively, and eluting separately the adsorbed enzyme inhibitors and enzymes from the columns to obtain fractions containing the physiologically active substances. The proteolytic enzyme may be kallikrein, plasmin, thrombin, trypsin, or chymotrypsin and the proteolytic enzyme inhibitor may be the corresponding inhibitor to these enzymes. Preferably the insolubilized enzyme/inhibitor system comprises the proteolytic enzyme inhibitor bound to an insoluble agarose, dextran or polyacrylamide carrier and the proteolytic enzyme chemically bound to an insoluble agarose, dextran or polyacrylamide carrier.