PURIFICATION AND CHARACTERIZATION OF ACIDIC PROTEASES FROM THE STOMACH OF THE DEEPWATER FINFISH ORANGE ROUGHY (HOPLOSTETHUS ATLANTICUS)

Abstract

Acidic proteases were extracted and purified from the stomach of orange roughy (Hoplostethus atlanticus). Protease I and II were glycoproteins with molecular weights of 33.5 and 34.5 KDa, respectively. Protease I had an isoelectric point of 5.30. The two forms of protease II (a and b) had isoelectric points of 4.35 and 4.40, respectively, and N-terminal sequence identity for 12 amino acids. The proteases exhibited optimal temperature activity at 37C. They had high activity at low temperatures and low thermal stability compared to mammalian pepsins. They were stable in the pH range of 2–4.5 and unstable above pH 6.5. Protease I and II had pH optima of 2.5 and 3.5, respectively, and K m’values for the hydrolysis of hemoglobin (pH 3.0, 37C) of 124 μM and 517 μM, respectively. Enzyme activities were inhibited by pepstatin A and high NaCl concentrations, and were slightly stimulated by Ca2+ and Cu2+.