Biochemical properties of pepsinogen and pepsin from the stomach of albacore tuna (Thunnus alalunga)

Pepsinogen (PG) from the stomach of albacore tuna (Thunnus alalunga) was purified to homogeneity by using a series of chromatographies involving Sephacryl S-200HR, Sephadex G-50 and DEAE-cellulose with a 658-fold increase in purity. Based on the native-PAGE and zymography, PG showed a single band with pepsin activity. Molecular weights (MW) of PG and active pepsin were estimated to be 39.9 and 32.7 kDa as determined by SDS–PAGE, respectively. PG was converted to the corresponding pepsin through an intermediate form (MW ≈ 36.8 kDa) and the complete activation was observed after 30–60 min. The N-terminal amino acid sequence of the first 15 amino acids of activation segment of pepsinogen was FHKLPLIKGKTAREE. The optimal pH and temperature for pepsin activity were 2.0 and 50 °C, respectively. The activity was stable in the pH range of 2–5. Residual activity more than 85% was found after heating at temperatures up to 50 °C for 30 min. Pepsin activity was strongly inhibited by pepstatin A, whilst E-64, ethylenediaminetetraacetic acid (EDTA) and soybean trypsin inhibitor exhibited the negligible effect. SDS and cysteine also showed inhibitory effects, whilst ATP, molybdate, NaCl and CaCl2 had no impact on pepsin activity.