[41] Cathepsin B, cathepsin H, and cathepsin L

This chapter describes two types of purification methods for Cathepsin B, Cathepsin H, and Cathepsin L. Method I is applicable to large amounts of frozen tissues, whereas method II is used with flesh tissue and takes advantage of a 50-fold purification factor attainable by isolation of lysosomes: it has the further advantage of separating the enzymes from inhibitors that are present in the cytosol and plasma. In first purification method, cathepsins B and H are purified from human liver. Method II involves purification of Cathepsins B, H, and L from rat liver. Method I include: extraction, autolysis, and acetone fractionation and DEAE-cellulose chromatography. The pool of cathepsin B from DEAE-cellulose is further purified by covalent chromatography on a column of aminophenylmercuric acetate coupled to Sepharose. Method II include: homogenization and cell fractionation gel; chromatography on Sephadex G-75; CM-Sephadex chromatography; chromatography of cathepsin L on concanavalin A-Sepharose. Cathepsin B can be with BZ-DL-Arg-NPhNO2 or Bz-Arg-2-NNap as substrate, wheras, Cathepsin H can be assayed selectively by use of an unblocked substrate such as Leu-NNap, Arg-NNap, or Arg-NMec. Three synthetic substrates have been used for cathepsin L assay: Bz-Arg-NH2, Z-Lys-OPhNO2, and Z-Phe-Arg-NMec.