New method to discriminate between cathepsin B and cathepsin L in crude extracts from fish muscle based on a simple acidification procedure

Abstract

Summary A new and simple method to distinguish between cathepsin B and cathepsin L in crude extracts of herring ( Clupea harengus ) muscle has been established. An acid treatment of crude extracts (exposed to pH 3 for 5 min) activated a latent form of cathepsin L and inactivated cathepsin B. Furthermore, in neutral crude extract, the hydrolysis of benzyloxycarbonyl‐ l ‐phenylalanyl‐ l ‐arginyl‐4‐methylcoumarine (Z‐Phe‐Arg‐MCA) (cathepsin B and cathepsin L substrates) was between 0% and 15% of the hydrolysis of benzyloxycarbonyl‐ l ‐arginyl‐ l ‐arginyl‐7‐amino‐4‐methylcoumarine (Z‐Arg‐Arg‐MCA; cathepsin B substrate). Cathepsin B activity is measured in neutral extract using the specific cathepsin B substrate Z‐Arg‐Arg‐MCA and cathepsin L activity is measured in acid‐treated extract with Z‐Phe‐Arg‐MCA as substrate. The specific cathepsin B inhibitor, CA‐074, did not inhibit the Z‐Arg‐Arg‐MCA significantly without affecting the Z‐Phe‐Arg‐MCA activity. An acid treatment of the crude extract is therefore a more advantageous approach to discriminate between cathepsin B and cathepsin L activities.