Dilution of digestive fish proteases with protein-free blocking reagents prevents loss of catalytic activity during microquantification

Abstract

The activity of proteases may be lost during microquantification because of their nonspecific binding to tubes and pipette tips. In this study, the effects of blocking reagents (blockers) in dilutions of digestive proteases of yellowtail, Seriola quinqueradiata, were compared. EzBlock Chemi (EBC; protein free), Western BLoT Blocking Buffer (WBB; protein free), casein-based blocker, and bovine serum albumin were evaluated by the measurement of trypsin, chymotrypsin, and aminopeptidase activities using fluorogenic substrates. Protease activities were linear in extracts of pyloric caeca at dilution rates of up to at least 1/6400 with the four blockers. However, the activities of pancreatic enzymes differed, and the highest levels were observed with EBC and WBB. Extracts diluted with EBC and WBB were incubated for up to 24 h at 25 °C. Pancreatic enzyme activities increased with time in extracts diluted with EBC, but for those diluted with WBB there was no clear trend. The activities of chymotrypsin and aminopeptidase in whole-body extracts of individual yellowtail larvae extracted with EBC were significantly higher than in those extracted with 150 mM NaCl, suggesting that EBC inhibits the loss of enzyme activity that may otherwise occur during extraction. Thus, EBC is considered to be the most effective diluent for the microquantification of proteases amongst the four blockers tested.