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 degrees 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 K(m)s of pepsins (P-I, P-II, P-III, and P-IV) for hemoglobin were calculated as 8.7 x 10(-8) M, 1.0 X 10(-7) M, 8.6 x 10(-8) M, and 7.3 x 10(-8) M, respectively. (c) 2006 Elsevier Ltd. All rights reserved.