Characterization of a stable intermediate in the unfolding of diazoacetylglycine ethyl ester?pepsin by urea

Abstract

The irreversible unfolding of covalently inhibited swine pepsin by urea was studied by spectrophotometric and viscosity measurements. At pH 4.5 and 25 °C in 8 M urea, a stable intermediate form of the protein was detected. It differed from the native protein by a slight loss of secondary structure and an increased intrinsic viscosity ([η] = 7.5 mL g−1), indicating the intermediate to have an increased molecular volume or to be more asymmetric in shape. The protein was transformed into a random coil form by increases of temperature and pH. Comparison with other results suggested that at pH 6 pepsin is less stable than its inactive precursor, pepsinogen, by about 3 Kcal mol−1 (1 cal = 4.187 J).