Analysis Note

Leupeptin gives multiple peaks on HPLC due to equilibria among three forms in solution. Purity determined using three main peaks. Majority of contaminating peptide is racemized leupeptin.

Application

Leupeptin has been used as a protease inhibitor: in ice-cold lysis buffer to harvest cells for western immunoblotting and immunoprecipitation,in chromatin immunoprecipitation (ChIP) lysis buffer for the isolation of fragmented chromatin samples in lysis buffer I to lyse the cells for tandem affinity purification (TAP)

Biochem/physiol Actions

Leupeptin helps to protect motoneurons from apoptosis. It also helps to enhance muscle function following nerve injury. It possesses neurotrophic action and has a growth-stimulating effect.

Inhibitor of serine and cysteine proteases. Inhibits plasmin, trypsin, papain, calpain, and cathepsin B. Does not inhibit pepsin, cathepsins A and D, thrombin, or α-chymotrypsin. Effective concentration 10-100 µM. There have been numerous studies using leupeptin to protect against hearing loss caused by acoustic overstimulation or the ototoxic antibiotic gentamicin. (Loss of cochlear hair cells is believed to be mediated by calpain.)