Stabilization of Conventional Cathepsin L Activity Assay in Crude Herring Muscle Extracts by NaCl Addition

The proteolytic activity of cathepsins plays a key role in post-mortem changes in fish muscle, affecting texture, flavour development and overall product quality. Reliable determination of cathepsin B and L activities in crude fish muscle extracts is challenging because of limited substrate specificity and strong matrix effects. The classical differential method is based on Z‑Arg‑Arg‑AMC (cathepsin B) and Z‑Phe‑Arg‑AMC (cathepsin B + L) with subsequent subtraction (L = (B + L) − B). However, differences in the catalytic efficiencies of cathepsin B against these two substrates and interference from other protease classes can lead to even negative calculated values for cathepsin L in crude extracts. In this study, extraction conditions and inhibitor profiling of purified cathepsins D, B and L from Baltic herring muscle were evaluated. The experiments revealed substantial interdependence between protease classes and demonstrated that the classical differential approach for cathepsin L determination may fail under crude‑extract conditions. Purified enzymes showed distinct NaCl sensitivities: cathepsins B and D were strongly inhibited at 3–5 g/100 g NaCl, whereas cathepsin L activity increased under the same conditions. These differences in salt sensitivity enabled the use of NaCl for functional enrichment of cathepsin L contribution to activity in crude extracts. The NaCl‑addition assay reduced non-specific protease contributions and eliminated subtraction-derived artefacts without the need for selective synthetic inhibitors. The proposed NaCl-addition approach retains the classical two-substrate framework while improving robustness and interpretability in complex fish muscle matrices. The approach can be readily adopted by laboratories without purchasing new substrates or inhibitors.