Denatured state of ovalbumin in high concentrations of urea as evaluated by disulfide rearrangement analysis.
To investigate the highly denatured state of ovalbumin (molecular mass of 42.7 kDa, four cysteine sulfhydryls and one cystine disulfide) using the disulfide rearrangement approach, we established the peptide-mapping procedure using a cysteine-labeling technique with a fluorescent dye that allows the quantitative analyses for the disulfide-involved half-cystines. Ovalbumin denatured at a low protein concentration in 8-10 M urea, in which the protein showed complete unfolding as evaluated by far-UV CD spectra, was analyzed for the disulfide-involved half-cystines using the peptide-mapping procedure. Data clearly showed that the number of free sulfhydryls and intrachain disulfides were four and one, respectively, but that all six half-cystines are labeled with the dye. These results strongly suggested that 15 disulfide isomers that are theoretically possible for a molecule having one disulfide and four sulfhydryls are all generated during the denaturation. The quantitative data for the ratios of the observed labeling values for the six half-cystines, relative to the overall labeling values, were consistent with the view that the distribution of the 15 possible disulfide isomers at equilibrium depends on the number of amino acid residues separating the two half-cystines to the power of -1.9 to -2.0. Essentially, the same non-gaussian chain nature was also observed with kinetic data for sulfhydryl-disulfide exchanges after denaturation of native ovalbumin.
Tatsumi
E
E
Research Institute for Food Science, Kyoto University, Japan.
Takahashi
N
N
Hirose
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003545:Cysteine; D004220:Disulfides / Q000032:analysis; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005456:Fluorescent Dyes; D007700:Kinetics; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D014508:Urea
D004220:Disulfides; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D014508:Urea; D010047:Ovalbumin; D003545:Cysteine; D004229:Dithiothreitol