Conformation of bilirubin oxidase in native and denatured states |
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Authors: | Tatsuya Samejima Chuen-Shang C Wu Kazunori Shiboya Hiroyuki Kaji Satoshi Koikeda Keiichi Ando and Jen Tsi Yang |
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Institution: | (1) Department of Chemistry, College of Science and Engineering, Aoyama Gakuin University, 157 Tokyo, Japan;(2) Cardiovascular Research Institute, University of California, 94143-0130 San Francisco, California;(3) Present address: Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, 192-03 Tokyo, Japan;(4) Tsukuba Research Laboratories, Amano Pharmaceutical Co., Tsukuba, 305 Ibaragi, Japan |
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Abstract: | The conformation of bilirubin oxidase (EC 1.3.3.5) fromMyrothecium 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 -sheets. There was another negative band at 187 nm that is absent from the spectra of model -helix or -sheet. CD analysis by the method of Changet 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 Garnieret al. AtpH 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°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°C. Thus, our denaturation studies suggest that bilirubin oxidase has a rigid tertiary structure. |
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Keywords: | Bilirubin oxidase circular dichroism conformation denaturation |
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