Radiation damage to a protein solution, detected by synchrotron X-ray small-angle scattering: dose-related considerations and suppression by cryoprotectants.
In small-angle X-ray scattering experiments at high-brilliant synchrotron sources, protein aggregation results from radiation damage. The radiation-induced aggregation of lysozyme in solution was qualitatively evaluated based on forward scattering and radii of gyration. The scattering did not change below 400 Gy and increased exponentially above this dose. The aggregation is only seen beyond the critical dose rate, and the 'dilution effect' known in radiology was also observed. Mass spectroscopy of the lysozyme solution exposed to a monochromatic X-ray beam did not show any cleavage of the polypeptide chain. Small-angle X-ray scattering patterns suggested that the radiation-induced aggregation should be a non-specific association of intact lysozyme, without substantial alterations of the folding topologies. It was found that the addition of small amounts of cryoprotectants, such as glycerol, ethylene glycol and sucrose, effectively reduced the radiation damage. Glycerol and ethylene glycol were identically effective in the 100 mM concentration range. A similar effective concentration was observed for sucrose. The damage reduction by the cryoprotectants was mainly ascribed to changes in the protein-protein interactions, and rarely to decreases in the diffusion rates of activated species.
Kuwamoto
Shigeo
S
Laboratory for Structural Biochemistry, RIKEN Harima Institute/SPring-8, Sayo, Hyogo 679-5148, Japan.
Akiyama
Shuji
S
Fujisawa
Tetsuro
T
D003451:Cryoprotective Agents / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D046912:Multiprotein Complexes / Q000032:analysis / Q000737:chemistry* / Q000528:radiation effects*; D009113:Muramidase / Q000032:analysis / Q000737:chemistry* / Q000528:radiation effects*; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects / Q000528:radiation effects; D011829:Radiation Dosage; D011836:Radiation Tolerance / Q000187:drug effects; D012996:Solutions; D017356:Synchrotrons; D014961:X-Ray Diffraction / Q000295:instrumentation / Q000379:methods*
D003451:Cryoprotective Agents; D046912:Multiprotein Complexes; D012996:Solutions; C421864:hen egg lysozyme; D009113:Muramidase