Purification and characterization of two pepsins from the stomach of pectoral rattail ( Coryphaenoides pectoralis)

Abstract

Two pepsins (A and B) were purified from the stomach of pectoral rattail ( Coryphaenoides pectoralis) by acidification, ammonium sulfate precipitation, gel filtration chromatography and anion exchange chromatography to obtain a single band on native-PAGE and SDS-PAGE. The purities of pepsin A and B were increased to 7.1- and 13.0-fold with approximately 5.7% and 2.2% yield, respectively. Pepsin A and B had the apparent molecular weights of 35 and 31 kDa, respectively, when analyzed using SDS-PAGE and Sephacryl S-200 gel filtration. Pepsin A and B showed maximal activity at pH 3.0 and 3.5, respectively, and had the same optimal temperature at 45 °C using hemoglobin as a substrate. Both pepsin A and B were stable in the pH range of 2.0–6.0 but were unstable at the temperatures greater than 40 °C. Activity of both pepsins was inhibited by pepstatin A and was activated by divalent cations, indicating pepsin characteristics. Activities of both pepsins continuously decreased as NaCl concentration increased (0–30%). The enzymes had high affinity and activity toward hemoglobin with K m and K cat values of 98–152 μM and 32–50 S − 1 , respectively. Purified pepsins generally showed the similar characteristics to other fish pepsins.