ION EXCHANGERS: A USEFUL TOOL FOR SEPARATION AND SIMULTANEOUS PURIFICATION OF LYSOSOMAL CYSTEINE PROTEINASES, CATHEPSINS B, H AND L.

Lysosomes of mammalian tissues contain a number of cysteine proteinases such as Cathepsins B, H and L, which participate in intracellular protein degradation and are involved in various pathological disorders. These proteinases have been purified to ~ 1600 fold with ~25% yields from goat liver. These Cathepsins having similar molecular weight (~25,000 Da) were obtained as a single pool after initial purification steps i.e. homogenization of acetone powder, acid autolysis at pH 4.0, 30-70 % (NH₄)₂SO₄ fractionation and molecular sieve chromatography on Sephadex G-100, were simultaneously separated and purified on exchange chromatographies. Cathepsin L was completely separated and purified from Cathepsin B & H at CM- Sephadex C-50 column at pH 5.6 and was eluted as bound protein at 0.62 M NaCl with a purification fold ~1629 and 22 % yield. Cathepsin B was eluted as bound protein in ~1545 fold with ~45% yield at 0.40 M concentration. Complete separation of Cathepsin H from Cathepsin B was achieved on DEAE-Sephadex A-50 at pH 6.0 where the former was obtained as unbound protein and the latter; Cathepsin H thus obtained was purified to ~ 1601.11 fold with ~29.18% yield. Cathepsin B, H & L were thus purified using ion-exchange chromatography.