Crude extract was obtained through homogenizing mackerel pyloric caeca with water, followed by the filtration of the homogenate through Celite. By precipitating the extract with ammonium sulfate under the saturation degree from 0.3 to 0.7, the proteinase fraction was isolated from the extract.
The fractionation of this precipitate by chromatography on DEAE-cellulose column caused it to be separated into six enzyme fractions, of which only one fraction having a high activity: the specific fraction being further purified by gel filtration with Sephadex G-200.
By this purification was made the removal of two sorts of contaminating proteins from the proteinase fraction. Concerning the structure and molecular size the enzyme preparation seemed to have been almost homogeneous.