Cathepsin D is a lysosomal aspartic protease of the pepsin family. Human cathepsin D is synthesized as a precursor protein, consisting of a signal peptide (residues 118), a propeptide (residues 1964), and a mature chain (residues 65412). The mature chain can be processed further to the light (residues 65161) and heavy (residues 169412) chains. It is expressed in most cells and overexpressed in breast cancer cells. It is a major enzyme in protein degradation in lysosomes, and also involved in the presentation of antigenic peptides. Mice deficient in this enzyme showed a progressive atrophy of the intestinal mucosa, a massive destruction of lymphoid organs, and a profound neuronal ceroid lipofucinosis, indicating that cathepsin D is essential for proteolysis of proteins regulating cell growth and tissue homeostasis. The activity of recombinant human CTSD is measured by its ability to cleave a fluorogenic peptide substrate MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2 in the assay buffer 0.1 M NaOAc, 0.2 M NaCl, pH 3.5. The rhCTSD is diluted to 20 ug/ml in assay buffer, then incubated at 37 °C for 30min. The incubated rhCTSD is diluted to 5 ug/mL in assay buffer. Loading into a black well plate 50 µL of 5 ug/mL rhCTSD and start the reaction by adding 50 µL of 60 µM substrate, with a substrate blank containing 50 µL assay buffer, 50 µL substrate, and no rhCTSD. Then read at excitiation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes. The specific activity of recombinant human CTSD is > 675 pmol/min/µg.