Purification and partial characterization of cathepsin D from porcine (Sus scrofa) liver using affinity chromatography

Abstract

Cathepsin D, a lysosomal aspartic protease, has been purified from porcine liver using a combination of pepstatin-A agarose and Affi-Gel Blue affinity chromatography, followed by size-exclusion chromatography. The purified protein consists of two polypeptide chains of 15 and 30 kDa, and has an isoelectric point of 6.8. Porcine liver cathepsin D has maximum activity at pH 2.5-3.0 as determined by its activity against hemoglobin, with a K(cat) of 14.3 s-1 and a k(cat)/K(M) of 2.70 x 106 s-1M-1 as determined by the hydrolysis of a fluorogenic peptide substrate.