Role of an intrachain disulfide bond in the conformation and stability of ovalbumin.
Ovalbumin, which contains one intrachain disulfide bond and four cysteine sulfhydryls, was reduced with dithiothreitol under non-denaturing conditions, and its conformation and stability were compared with those of the disulfide-bonded form. The CD spectrum in the far-UV region revealed that the overall conformation of the reduced form is similar to that of the disulfide-bonded one. Likewise, the inaccessibility to trypsin and the non-reactivity of the four cysteine sulfhydryls, exhibited by the native disulfide-bonded ovalbumin, were still retained in the disulfide-reduced form. Thus, the reduced ovalbumin appeared to substantially take the native-like conformation. However, the near-UV CD spectrum slightly differed between the native and disulfide-reduced forms. Protein alkylation with a fluorescent dye and subsequent sequence analysis showed that the two sulfhydryls (Cys73 and Cys120) originating from the disulfide bond are highly reactive in the reduced form. Furthermore, upon proteolysis with subtilisin, the N-terminal side of Cys73 was cleaved in the reduced form, but not in the disulfide-bonded one. Upon heat denaturation, the transition temperature of the reduced form was lower, by 6.8 degrees C, than that of the disulfide-bonded one. Thus, we concluded that ovalbumin has a native-like conformation in its disulfide-reduced form, but that the local conformation of the reduced form fluctuates more than that of the disulfide-bonded one. Such local destabilization may be related to the decreased stability against heat denaturation.
Takahashi
N
N
Research Institute for Food Science, Kyoto University.
Koseki
T
T
Doi
E
E
Hirose
M
M
D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003545:Cysteine / Q000032:analysis; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D006358:Hot Temperature; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000737:chemistry*; D010084:Oxidation-Reduction; D010455:Peptides / Q000302:isolation & purification; D011487:Protein Conformation; D013381:Subtilisins / Q000737:chemistry; D014357:Trypsin
D004220:Disulfides; D010455:Peptides; D010047:Ovalbumin; D010450:Endopeptidases; D013381:Subtilisins; D014357:Trypsin; D003545:Cysteine