Catalytic and physico-chemical characteristics of goat spleen cathepsin B

To improve the level of purity of cathepsin B, we have modified the published procedure [Agarwal, S.K. and Khan, M.Y. (1987) Biochem. Int. 15, 785-792] by incorporating CM-Sephadex ion exchange chromatography and chromatofocusing. The enzyme thus isolated could be resolved into one 26 kDa major and a miner 2.7 kDa protein bands on SDS-PAGE. The two components, however, could not be separated by gel filtration and they eluted, in a single peak corresponding to a molecular mass of 28.1 kDa. Among the various substrates tested, Z-Phe-Arg-MCA with a K-m of 0.058 mM and hemoglobin with a K-m of 1.449 mu M were the most preferred synthetic and protein substrates respectively. It was found to be a glycoprotein with an acidic pi of 4.8. The enzyme was activated by various thiol-reducing reagents and inhibited by cysteine proteinase inhibitors, divalent cations, lysyl group modifiers, anti-inflammatory drug and denaturing agents. The hydrodynamic behaviour of cathepsin B suggested a compact and globular conformation. Immunodiffusion studies with anti-goat cathepsin B indicated a tissue/species dependence.