Purification and characterization of two pepsins from the stomach of pectoral rattail (Coryphaenoides pectoralis).
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 degrees 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 degrees 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 Km and Kcat values of 98-152 microM and 32-50 S(-1), respectively. Purified pepsins generally showed the similar characteristics to other fish pepsins.
Klomklao
Sappasith
S
Department of Food Science and Technology, Faculty of Technology and Community Development, Thaksin University, Phattalung Campus, Phattalung, 93110, Thailand. sappasith@tsu.ac.th
Kishimura
Hideki
H
Yabe
Mamoru
M
Benjakul
Soottawat
S
D000818:Animals; D048388:Gadiformes; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010434:Pepsin A / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011489:Protein Denaturation / Q000187:drug effects; D012965:Sodium Chloride / Q000494:pharmacology; D013270:Stomach / Q000737:chemistry*; D013696:Temperature
D012965:Sodium Chloride; D010434:Pepsin A; C095924:pepsin B