Purification and characterization of pepsinogen and pepsin from the stomach of rainbow trout (Oncorhynchus mykiss)

Abstract

Three pepsinogen isolates (PG-I till III) from the stomach of rainbow trout (Oncorhynchus mykiss) were purified by using ammonium sulfate precipitation, ion exchange chromatography, and two subsequent gel filtrations containing seven pepsinogen isoforms. SDS-PAGE revealed that pepsinogen isolate I contained two isoforms with molecular masses of 45 kDa (PG-I a) and 44 kDa (PG-I b) and the molecular masses of the PG isolates PG-II and PG-III were 42 kDa, respectively. P-I till P-III converted into the corresponding pepsins (P) at pH 2.0 with molecular masses of 37 kDa (P-I a, b, P-III) and 35 kDa (P-II). The isoelectric points were 4.0 (PG-I a, b), 5.9 (PG-II), and pepsinogen isolate III exhibited four isoforms with isoelectric points of 3.73 (PG-III a), 3.78 (PG-III b), 4.0 (PG-III c) and 4.15 (PG-III d). After conversion from pepsinogen, trout pepsins exhibited optimal activity at pH 3.0 and 40 °C (P-I) and pH 2.5 and 30 °C (P-II, P-III). The N-terminal amino acid sequences of the three pepsin isolates (P-I till III) were determined up to 20 amino acids. P-II showed 100 % identity (ID) to pepsin A of Oncorhynchus keta, but P-I and P-III revealed high similarity to chitinases (85 % ID). Catalytic constants KM and kcat for proteolysis of acid-denatured hemoglobin were determined as 2.8 × 10−8 M (P-I), 1.3 × 10−8 M (P-II) and 7.9 × 10−9 M (P-III), and 5.43–19.1 S−1, respectively.