Purification and characterization of cathepsin B from buffalo (Bubalus bubalis) lung

In an attempt to improve the level of purity of buffalo lung cathepsin B, we have modified the earlier procedure by incorporating CM- Sephadex C-50 ion exchange chromatography and rechromatography on Sephacryl S-200 column. Appearance of a single band on SDS-PAGE under reducing and non-reducing conditions showed that the enzyme exists in a single chain molecule. Molecular weight of purified cathepsin B was determined to be 23800 and 25400 by SDS-PAGE and gel filtration, respectively. The enzyme, a glycoprotein, showed activity against α-N-benzoyl-DL-arginine-2-naphthylamide and α-N-benzoyl-DL-arginine-4-nitroanilide. The physico-chemical properties of the enzyme were similar to the properties reported for cathepsin B from other sources. However, the NH2-terminal amino acid residue of the enzyme was found to be Ala as against Leu reported from other sources. The enzyme was activated by various thiol reducing reagents and inhibited by cysteine protease inhibitors and denaturing agents. The hydrodynamic behaviour of cathepsin B which includes Stokes radius (2.29 nm), frictional ratio (1.19) and intrinsic viscosity (3.08 ml/gm) suggested that the native enzyme conformation is compact and globular.