Ostrich pepsins I and II: A kinetic and thermodynamic investigation

Abstract

Two forms of ostrich pepsin have been purified, ostrich pepsins I and II. The aim of this study was to characterize these pepsins in terms of temperature and alkaline stability, temperature and pH optima, and to investigate their kinetic properties. The effect of pH and temperature on ostrich pepsins was studied using the haemoglobin assay method. The hydrolysis of a synthetic hexapeptide substrate Leu-Ser-Phe-(NO 2 )-Nle-Ala-Leu-OMe was followed spectrophotometrically at 310 nm. The activity of pepsins towards short synthetic substrates were investigated using ninhydrin. Inhibition studies were performed with the reversible inhibitor pepstatin A and the inhibitors DAN, EPNP and p -bromophenacyl bromide. Ostrich pepsins exhibited an optimum pH of 2.0 towards haemoglobin, and were stable up to pH 7. 5. The optimum temperature for ostrich pepsin II was at 60°C, while ostrich pepsin I showed a broader optimum temperature range (40–60°C). Ostrich pepsin I was more susceptible to heat inactivation than ostrich pepsin II. Both pepsins showed a lower activity towards haemoglobin than porcine pepsin. Only ostrich pepsin II could hydrolyse the hexapeptide substrate, albeit at a much slower rate than porcine pepsin. The activity of the ostrich pepsins towards short synthetic peptides was generally very low. Pepstatin A is a very potent inhibitor of ostrich pepsins with a K i of 2.14–2.2 × 10 −8 M. EPNP, DAN and p -bromophenacyl bromide were all inhibitory to ostrich pepsin II, DAN requiring the presence of Cu 2+ . Ostrich pepsins exhibited similar thermal and kinetic properties when compared to pepsins of avian origin. Avian pepsins are generally more stable to alkaline conditions, and exhibit low activity towards short synthetic substrates.