AMCA-Glu-Glu-Lys-Pro-Ile-Ser-Phe-Phe-Arg-Leu-Gly-Lys(biotinyl)-NH₂

Specific substrate for cathepsin D (Km = 53 µM, kcat = 7.28 s?¹, kcat/Km = 0.14 µM?¹s?¹) with a single cathepsin cleavage site between the two Phe residues. It is flanked by an N-terminal AMCA-fluorophore (7-amino-4-methylcoumarin-3-acetic acid) and a C-terminal biotin moiety. After enzymatic hydrolysis the biotin-conjugated cleavage product and undigested substrate AMCA-EEKPISFFRLGK(Biotinyl)-amide can be removed with streptavidin-coated magnetic beads. The remaining fluorescent peptide in solution represents the amount of digested substrate. The versatility of this cathepsin D 'digest and pull down assay' is applicable for routine assays.