ABSTRACT
The irreversible unfolding of covalently inhibited swine pepsin by urea was studied by spectrophotometric and viscosity measurements. At pH 4.5 and 25 degrees 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 ([pi] = 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).
Subject(s)
Azo Compounds , Glycine , Pepsin A/analogs & derivatives , Urea/pharmacology , Azo Compounds/analogs & derivatives , Circular Dichroism , Glycine/analogs & derivatives , Hot Temperature , Hydrogen-Ion Concentration , Pepsinogens , Protein Conformation/drug effects , Protein DenaturationABSTRACT
By modifying four tyrosine residues in pepsin a derivative (aminopepsin) is obtained which is capable to conjugate with a fluorescent label DNS-Cl without loss of the catalytic activity. Rotational relaxation times of native pepsin and dansylated aminopepsin (DAP) are measured by a fluoresence polarization method. The values obtained are shown to be lower than those calculated for arigid pepsin globule. Possible sources of the obtained difference are discussed. A reversible or covalent blocking of pepsin or DAP active centres by specific inhibitors leads to an increase in rotational relaxation time values for these proteins reaching the magnitude which is very close to that calculated for a model of rigid pepsin. Brownian relaxation of pepsin and DAP reduced by beta-mercaptoethanol and of pepsinogen and some fragments of pepsine macromolecule in aqueous solutions is invigated as well. The results are intepreted as representing an intramolecular mobility or segmental flexibility of pepsin and DAP. With the use of the obtained and X-ray data a segmental model of dynamic pepsin structure is suggested. On the basis of this model some conclusions are drawn concerning a localization of a polypeptide which is split off from the N-terminus of pepsinogen during its activation. A possible role of segmental flexibility in the catalytic action of pepsin is considered.
