Purification and characterization of sea bream (Sparus latus Houttuyn) pepsinogens and pepsins

Four pepsinogens (PG-I, PG-II, PG-III, and PG-IV) were identified in sea bream (Sparus latus Houttuyn) stomach and were purified to homogeneity by 20–60% ammonium sulfate fractionation and several chromatographies. The molecular weights of the four purified PGs were 36, 32, 32, and 34 kDa, respectively. All the pepsinogens converted into pepsins within a few minutes under pH 2.0. The molecular weights of all the four pepsins were approximately 30 kDa as determined by SDS–PAGE. Optimum pHs of the four enzymes were 3.0–3.5, and optimum temperatures were 45–50 °C. Western blot analysis revealed that anti-PG-II antibody cross-reacted with all the four pepsins while scarcely any immunological cross-reaction existed between anti-PG-I antibody and other pepsins. From the Lineweaver–Burk double reciprocal plots, the Kms of pepsins (P-I, P-II, P-III, and P-IV) for hemoglobin were calculated as 8.7 × 10−8 M, 1.0 × 10−7 M, 8.6 × 10−8 M, and 7.3 × 10−8 M, respectively.