Identification of pepsinogens and pepsins from the stomach of European eel (Anguilla anguilla)

Three pepsinogens (PG-I, PG-II and PG-III) were purified to homogeneity from the stomach of freshwater fish European eel (Anguilla anguilla) by ammonium sulphate fractionation, column chromatography on anion exchange and gel-filtration. Their molecular masses were determined as 36, 37 and 36 kDa, respectively, by SDS–PAGE, which were in agreement with the results obtained by Sephacryl S-200 and Superdex 75 gel-filtration. PG-I, PG-II and PG-III converted into corresponding pepsins with molecular masses of approximately 30 kDa at pH 2.0 and showed maximal activity at pH 3.5, 2.5 and 2.5. Optimal temperatures of these pepsins were 40, 40 and 35 °C, using bovine haemoglobin as substrate. Western blot analysis revealed that anti-sea bream PG-I and PG-II polyclonal antibodies cross-reacted with all three PGs of European eel. On the other hand, anti-sea bream PG-III and PG-IV antibodies cross-reacted with PG-II and PG-III of European eel, while no cross-reaction with PG-I was detected. The kinetic constants of Km and kcat of pepsins (P-I, P-II and P-III) for haemoglobin were calculated as 8.8 × 10−5 M, 23.7 s−1; 9.2 × 10−5 M, 19.4 s−1 and 7.0 × 10−5 M, 34.4 s−1, respectively.