Structural modifications of human beta 2 microglobulin treated with oxygen-derived radicals.
Treatment of human beta 2 microglobulin (beta 2m) with defined oxygen-derived species generated by treatment with gamma-radiation was studied. As assessed by SDS/PAGE, the hydroxyl radicals (.OH) caused the disappearance of the protein band at 12 kDa that represents beta 2m, and cross-linked the protein into protein bands stable to both SDS and reducing conditions. However, when .OH was generated under oxygen in equimolar combination with the superoxide anion radical (O2.-), the high-molecular-mass protein products were less represented, and fragmented derivatives were not obviously detectable. Exposure to .OH alone, or to .OH + O2.- in the presence of O2, induced the formation of beta 2m protein derivatives with a more acidic net electrical charge than the parent molecule. In contrast, O2.- alone had virtually no effect on molecular mass or pI. Changes in u.v. fluorescence during .OH attack indicated changes in conformation, as confirmed by c.d. spectrometry. A high concentration of radicals caused the disappearance of the beta-pleated sheet structure and the formation of a random coil structure. Loss of tryptophan and significant production of dityrosine (2,2'-biphenol type) were noted, exhibiting a clear dose-dependence with .OH alone or with .OH + O2.-. The combination of .OH + O2.- induced a pattern of changes similar to that with .OH alone, but more extensive for c.d. and tryptophan oxidation (2 Trp/beta 2m molecule), and more limited for dityrosine formation. Lower levels of these oxidative agents caused the reproducible formation of species at 18 and 25 kDa which were recognized by antibodies against native beta 2m. These findings provide a model for the protein pattern observed in beta 2m amyloidosis described in the literature.
Capeillere-Blandin
C
C
INSERM U25 et CNRS URA 122, Clinique Néphrologique, Hôpital Necker, Paris, France.
Delaveau
T
T
Descamps-Latscha
B
B
D000596:Amino Acids / Q000032:analysis; D002942:Circular Dichroism; D005609:Free Radicals; D005720:Gamma Rays; D006801:Humans; D006878:Hydroxides / Q000494:pharmacology*; D017665:Hydroxyl Radical; D007700:Kinetics; D011487:Protein Conformation / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013481:Superoxides / Q000494:pharmacology*; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000187:drug effects / Q000528:radiation effects
D000596:Amino Acids; D005609:Free Radicals; D006878:Hydroxides; D001613:beta 2-Microglobulin; D013481:Superoxides; D017665:Hydroxyl Radical