Protease activities in raw milk determined using a synthetic heptapeptide substrate

A synthetic heptapeptide (H-Pro-Thr-Glu-Phe-[p-nitro-Phe]Arg-Leu-OH) was us ed as substrate for detection and assay of cathepsin D in raw bovine milk. Cathepsin D produced a specific peptide, as detected by HPLC analysis of pe aks for product and substrate. On incubation of acid wheys from milk sample s with the substrate, three hydrolysis products were detected and bonds cle aved were identified by mass spectrometry. Inhibition studies were performe d to identify enzymes responsible for the hydrolysis. One activity was cath epsin D and production of another peptide was inhibited by cysteine proteas e inhibitors, suggesting cysteine protease activity in milk.