Highly ordered molten globule-like state of ovalbumin at acidic pH: native-like fragmentation by protease and selective modification of Cys367 with dithiodipyridine.

Structural characteristics of ovalbumin at acidic pH were investigated by a variety of analytical approaches. At pH 2.2, the protein appeared to assume a partially denatured, molten globule-like conformation as evaluated by the binding of a hydrophobic probe, anilino-1-naphthalene-8-sulfonate. The protein was, however, resistant to proteolysis with pepsin under conditions in which the urea-denatured form was extensively hydrolyzed. Furthermore, under more drastic proteolytic conditions, the acid ovalbumin was specifically proteolyzed at the N-terminal site of Ala351, which is located in close proximity to the canonical serpin cleavage site Ala352-Ser353 that is known to be the cleavage site at neutral pH with subtilisin and elastase in native ovalbumin. Among the four cysteine residues (Cys11, Cys30, Cys367, and Cys382), which are all known to be buried in the native ovalbumin molecule, only Cys367 was specifically modified with 2,2'-dithiodipyridine, generating a mixed-disulfide protein derivative. Upon incubation of the derivative with a high concentration of L-cysteine, the thiopyridine mixed disulfide did not undergo any bimolecular exchange reaction with the thiol in the absence of an added denaturant, indicating that the mixed disulfide group is inaccessible. The far-UV CD spectra indicated that the native secondary structure is retained in either the modified or non-modified protein; but as evaluated by the near-UV CD spectra, the asymmetric nature of aromatic side chains in the non-modified ovalbumin and of the mixed-disulfide group in the modified protein was almost lost at pH 2.2. These results are consistent with a highly ordered molten globule-like state for OVA at pH 2.2, in which side chains, but not the backbone chain, significantly fluctuate.