Conformation of bilirubin oxidase in native and denatured states.
The conformation of bilirubin oxidase (EC 1.3.3.5) from Myrothecium verrucaria was studied by circular dichroism (CD). The far-UV CD spectrum showed a single minimum at 215 nm and a maximum near 198 nm, suggesting the dominance of beta-sheets. There was another negative band at 187 nm that is absent from the spectra of model alpha-helix or beta-sheet. CD analysis by the method of Chang et al. agreed well with the estimates based on the Chou and Fasman sequence-predictive method, but the Provencher-Glöckner method of CD analysis agreed well with the sequence-predictive method of Garnier et al. At pH 12 the 215- and 187-nm bands completely disappeared and the protein was denatured. This denaturation was accompanied by the appearance of a large positive band at 250 nm, probably due to ionization of tyrosine residues. In 20 mM sodium dodecyl sulfate the magnitude of the 215-nm band increased, but the spectrum transformed to that of partial helices after heating at 100 degrees C. In 6 M guanidine hydrochloride the far-UV CD spectrum was monotonic and became more negative at the lower wavelength limit (near 212 nm), suggesting that the secondary structure of the protein was disrupted. However, the near-UV CD spectrum retained residual aromatic bands even after heating at 100 degrees C. Thus, our denaturation studies suggest that bilirubin oxidase has a rigid tertiary structure.
Samejima
T
T
Department of Chemistry, College of Science and Engineering, Aoyama Gakuin University, Tokyo, Japan.
Wu
C S
CS
Shiboya
K
K
Kaji
H
H
Koikeda
S
S
Ando
K
K
Yang
J T
JT
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D019791:Guanidine; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017421:Sequence Analysis / Q000379:methods; D012967:Sodium Dodecyl Sulfate; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet
D006146:Guanidines; D012996:Solutions; D012967:Sodium Dodecyl Sulfate; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C042401:bilirubin oxidase; D019791:Guanidine