Conformational state of disulfide-reduced ovalbumin at acidic pH.
Ovalbumin assumes a highly ordered molten-globule conformation at pH 2.2. To investigate whether or not such structural nature is related to the existence of an intrachain native disulfide bond, the structural characteristics of disulfide-reduced ovalbumin at the acidic pH were compared with those of the native disulfide-intact protein by a variety of analytical approaches. The disulfide-reduced protein was found to assume a partially denatured molten globule-like conformation similar to the disulfide-intact counterpart as analyzed by the CD and intrinsic tryptophan fluorescence spectra and by the binding of a hydrophobic probe of anilino-1-naphthalene-8-sulfonate. The results from size-exclusion chromatography also showed that the disulfide-reduced and disulfide-intact proteins have essentially the same compact, native-like hydrodynamic volume. The disulfide-reduced protein was, however, highly sensitive to proteolysis by pepsin at the acidic pH under the proteolytic conditions in which the disulfide-intact protein was almost completely resistant. Furthermore, on a differential scanning calorimeter analysis the disulfide-reduced protein had an endothermic transition at a much lower temperature (Tm = 48.5 degrees C) than the disulfide-intact protein (Tm = 57.2 degrees C). Taken together, we concluded that the intrachain disulfide bond should not be directly related to the highly ordered molten-globule conformation of ovalbumin, but that its conformational stability depends on the presence of the disulfide bond.
Tatsumi
E
E
Research Institute for Food Science, Kyoto University, Japan.
Yoshimatsu
D
D
Hirose
M
M
D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010047:Ovalbumin / Q000737:chemistry*; D010084:Oxidation-Reduction; D010434:Pepsin A / Q000737:chemistry; D011487:Protein Conformation; D014364:Tryptophan / Q000737:chemistry
D004220:Disulfides; D014364:Tryptophan; D010047:Ovalbumin; D010434:Pepsin A