THE PURIFICATION OF CATHEPSIN

1. One mg. of the purified cathepsin whose preparation is described is as active as the extract of 1.3 gm. of spleen. An eightfold further purification is possible by procedures which are still being modified and so are not described in the present paper. 2. The first step of the purification consists in suspending frozen and thawed spleen in water and letting it autolyze. 3. In the second step, ammonium sulfate is added and the suspension is acidified and warmed. Much additional autolysis takes place. The cathepsin is protected from destruction by being adsorbed to the insoluble spleen material. When this insoluble material is filtered off most of the products of autolysis remain in the filtrate. 4. The cathepsin is then released from the insoluble spleen material by making the suspension slightly alkaline. Some inert protein still remains adsorbed to the insoluble spleen material. 5. More inert protein is removed by adsorption with aluminum hydroxide formed in the cathepsin solution by the addition first of aluminum chloride and then of sodium hydroxide. Preformed aluminum hydroxide is a much less effective adsorbent. 6. The purified cathepsin is precipitated from very dilute solution with tungstic acid. Tungstic acid precipitates most proteins in the native form provided the solution is not too acid. 7. Further evidence is given that cathepsin is not a proteinase of the papain type.