| The characterization of fish pepsin is an important part of fish physiological biochemistry research, and is also the rationale of fish feed development. So the study of fish pepsin will be beneficial to feed development.Four pepsinogens have been purified from sea bream and mandarin fish, respectively by ammonium sulfate fractionation, DEAE-Sephacel chromatography and Sephacryl S-200 gel-filtration for the first time and their characteristics be studied. These study are not only the basis of the pepsin exploitaion but also helpful for physiological biochemistry research of these two economically fish.The molecular weights of the four purified sea bream PGs were 36, 32, 32 and 34 kDa, and those of mandarin fish PGs were about 36, 35, 38 and 34 kDa, respectively. All the pepsinogens converted into pepsins within a few minutes under pH 2.0. The molecular weights of eight pepsins were approximately 30 kDa as determined by SDS-PAGE.Using bovine hemoglobin as substrate, Optimum pHs were 3.0-3.5, and optimum temperatures were 45-50℃for sea bream pepsins. But the optimum pHs of the mandarin fish pepsins were 3.5, and optimum temperatures were 40-45℃.All these pepsins are aspartic proteinases, they can be inhibited by pepstatin in different molar ratios effectively. From the Lineweaver-Burk double reciprocal plots, the Kms of sea brem pepsins (P-I, P-II, P-III, P-IV) for hemoglobin were 8.7×10-8 mol/L, 1.0×10-7 mol/L, 8.6×10-8 mol/L and 7.3×10-8 mol/L, respectively. The Kms of the four mandarin fish pepsins were 2.3×10-7,1.2×10-7,2.4×10-8 and 1.3×10-7 mol/L .Western blot analysis revealed that anti-sea bream PG-II polyclonal antibody cross reacted positively with all the four pepsins from sea bream, while only weak immunological cross-reaction between anti-sea bream PG-I antibody with other pepsins was identified. Comparing the N-terminal amino acid sequences of PG-I and PG-II of mandarin fish, their identity reached 92%. |
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