Circular dichroic study of conformational changes in ovalbumin induced by modification of sulfhydryl groups and disulfide reduction.
Sulfhydryl groups of ovalbumin were chemically modified under denaturing conditions in the absence and presence of dithiothreitol, and effects on the secondary structure of the protein were investigated by circular dichroic (CD) measurements. The contents of alpha-helix, beta-structure, and "random coil" (unordered, nonrepetitive structure) were estimated by simulation of the CD spectra and using the parameters established by Chen et al. The principal findings were these: (1) Modification of the four free sulfhydryl groups [with 5,5'-dithiobis(2-nitrobenzoic acid), iodoacetate, or iodoacetamide] caused ovalbumin molecule to unfold partially and to undergo primarily helix-to-beta structure transition. (2) Cleavage of the disulfide bond did not lead to a further conformational change in the sulfhydryl-modified ovalbumin. (3) The remaining helical structure existed in a destabilized state with increased chain flexibility, as the modified protein was very susceptible to denaturation by guanidine and urea. (4) Further evidence for increased chain flexibility was provided by the finding that sodium dodecyl sulfate (SDS) induced helix formation in the sulfhydryl-modified, but not native, ovalbumin. And (5), since both nonreduced and reduced proteins, with their sulfhydryl groups blocked, displayed similar transitions in solutions of guanidine, urea, and SDS suggested that the single disulfide bond did not physically constrain the ovalbumin molecule.
Batra
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Department of Biochemistry, Wright State University, Dayton, Ohio 45435.
Sasa
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Ueki
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Takeda
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D002942:Circular Dichroism; D004220:Disulfides; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D010047:Ovalbumin / Q000648:ultrastructure*; D010084:Oxidation-Reduction; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013438:Sulfhydryl Compounds; D014508:Urea / Q000494:pharmacology
D004220:Disulfides; D006146:Guanidines; D013438:Sulfhydryl Compounds; D012967:Sodium Dodecyl Sulfate; D014508:Urea; D010047:Ovalbumin; D019791:Guanidine