Purification and characterization of chymotrypsins from the hepatopancreas of crucian carp (Carassius auratus)

Two chymotrypsins (chymotrypsin A and B) have been purified to homogeneity from the hepatopancreas of crucian carp (Carassius auratus) by ammonium sulphate fractionation and chromatographies on DEAE-Sepharose, Sephacryl S-200 HR, Phenyl-Sepharose and SP-Sepharose. The molecular masses of chymotrypsin A and B were approximately 28 and 27 kDa, respectively, by SDS–PAGE. Purified chymotrypsins also revealed single bands by native-PAGE. Optimum temperatures of chymotrypsin A and B were 40 and 50 °C, and optimal pHs were 7.5 and 8.0 using Suc-Leu-Leu-Val-Tyr-AMC as substrate. Both enzymes were effectively inhibited by serine proteinase inhibitors and slightly activated by metal ions such as Ca2+ and Mg2+, while inactivated by Mn2+, Cd2+, Cu2+, Fe2+ to different degrees. Apparent Kms of chymotrypsin A and B were 1.4 and 0.5 μM, and Kcats were 2.7 S−1 and 3.4 S−1, respectively. Immunoblotting analysis using anti-chymotrypsin B weakly cross reacted with chymotrypsin A.