| Pepsin is a member of the large family of aspartic proteinases, which hydrolyze proteins and peptides at the carboxyl side of hydrophobic and aromatic amino acid residues and play a critical role physiologically and in the digestion of foods. As a proteinase which reveals higher acitivy at low temperatures, pepsin has also been used in food processing and in the configuration of fish feeds. However, till now,very little information regarding pepsins from fresh water fish has been reported. In order to study the physiological and digestive functions of pepsins from fresh water fish, we purified and characterized pepsins from the stomach of rice field eel(Monopterus albus Zuiew)and European eel (Anguilla anguilla). Such a detailed study of pepsins will benefit not only for the manufacture of fish feeds and the development of aquaculture but also for the effective utilization of aquatic processing byproducts.Four pepsinogens were isolated from rice field eel and three pepsiogens were purified to homogeneity from the stomach of European eel by ammonium sulfate fractionation, column chromatographies on anion exchange and gel-filtration. Then these pepsiogens and the corresponding pepsins are ex??ive studied, which is not only the basis of the pepsin exploitation but also benefit for physiological biochemistry research of these two economically fish.The molecular masses of the four purified rice field eel pepsiogens were determined as 36, 36, 36 and 35 kDa, and those European eel pepsiogens were determined as 36, 37 and 36 kDa, respectively by SDS-PAGE. All the PGs converted into their corresponding active form pepsins (Ps) within few minutes at pH 2.0, and their activities could be completely suppressed by a typical asparatic proteinase inhibitor pepstatin A.Optimum pH of the four rice field eel pepsins and three European eel pepsins against hemoglobin were 3.5,3.0,3.0, 3.0, 3.5, 2.5, 2.5; Optimum temperature of the four rice field eel pepsins and three European eel pepsins against hemoglobin were 40℃, 40℃, 35℃, 35℃,40℃, 40℃, 35℃, respectively.The kinetic constant Km of these four rice field eel pepsins (PG-I, PG-II, PG-III(A) and PG-III(B)) for hemoglobin were 1.2×10-4 M, 8.7×10-5 M, 6.9×10-5 and 8.8×10-5 M, respectively. The turnover number (kcat) of them were 23.2, 24.0, 42.6 and 25.9 (s-1); the kcat/Km were 1.9×105 M-1·s-1, 2.8×105 M-1·s-1, 6.1×105 M-1·s-1 and 3.0×105 M-1·s-1. The kinetic constants of Km, kcat and kcat/Km of European eel pepsins (P-I, P-II and P-III) for hemoglobin were calculated as 8.8×10-5 M, 23.7 s-1, 2.7×105 M-1·s-1; 9.2×10-5 M, 19.4 s-1,2.1×105 M-1·s-1and 7.0×10-5 M, 34.4 s-1, 4.9×105 M-1·s-1, respectively.Immunological assay revealed that all the four rice field eel PGs positively reacted with anti-sea bream PG-I and PG-II polyclonal antibodies. Anti-sea bream PG-III antibody positively reacted with rice field eel PG-III(A) and PG-III(B) while no cross reaction toward rice field eel PG-I and PG-II could be detected. Similarly, anti-sea bream PG-I and PG-II polyclonal antibodies cross reacted with 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 to PG-I was detected. |
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