Protein damage, induced by small amounts of photodynamically generated singlet oxygen or hydroxyl radicals.
The influence of limited oxidation of glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12), alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) and myoglobin by singlet oxygen and by hydroxyl radicals was investigated. The intrinsic fluorescence of glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase decreased rapidly during oxidation, indicating a conformational change of the protein molecules. The free energy of isothermal unfolding in urea solutions was increased by singlet oxygen, but decreased by hydroxyl radical attack. The velocity of refolding of the denatured protein after dilution of the denaturant was increased by exposure to either singlet oxygen or hydroxyl radicals, with one exception: the velocity of refolding of myoglobin, oxidized by singlet oxygen, was strongly decreased. Hydroxyl radicals produced covalently crosslinked protein aggregates and some fragmentation, whereas singlet oxygen produced only crosslinked aggregates with glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase. All oxidized proteins were more susceptible to proteolysis by elastase and proteinase K, as compared to the undamaged proteins. Singlet oxygen-induced crosslinked aggregates were degraded very rapidly by elastase. Hydroxyl radical-induced aggregates of glyceraldehyde-3-phosphate dehydrogenase were also degraded very rapidly by this enzyme, but hydroxyl radical-induced aggregates of alcohol dehydrogenase were resistent to enzymatic degradation. The results indicate that limited protein oxidation may have a pronounced influence on several properties of the protein. The effects vary, however, with varying proteins and with the oxidizing species.
Prinsze
C
C
Sylvius Laboratories, Department of Medical Biochemistry, Leiden, The Netherlands.
Dubbelman
T M
TM
Van Steveninck
J
J
D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000818:Animals; D005609:Free Radicals; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006868:Hydrolysis; D006878:Hydroxides / Q000378:metabolism*; D017665:Hydroxyl Radical; D009211:Myoglobin / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D010196:Pancreatic Elastase; D010777:Photochemistry; D011489:Protein Denaturation
D005609:Free Radicals; D006878:Hydroxides; D009211:Myoglobin; D017665:Hydroxyl Radical; D000426:Alcohol Dehydrogenase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010196:Pancreatic Elastase; D010100:Oxygen