A comparison of quantitative and qualitative superoxide dismutase assays for application to low temperature microalgae
| Autor: | T. S. Gechev, van de Willem Poll, Paul J. Janknegt, Anita G. J. Buma, Jan W. Rijstenbil |
|---|---|
| Přispěvatelé: | Ocean Ecosystems, Marine Microbiology |
| Jazyk: | angličtina |
| Rok vydání: | 2007 |
| Předmět: |
Antioxidant
medicine.medical_treatment Sonication UV-B RADIATION Biophysics native gel electrophoresis TETRASELMIS-GRACILIS PRASINOPHYCEAE CHILLING STRESS Buffers xanthine/xanthine oxidase assay MARINE MACROALGAE Superoxide dismutase chemistry.chemical_compound Protein purification Methods medicine Radiology Nuclear Medicine and imaging OXIDATIVE STRESS OZONE DEPLETION Xanthine oxidase MOLECULAR-OXYGEN chemistry.chemical_classification Reactive oxygen species Radiation Chromatography Radiological and Ultrasound Technology biology Extraction (chemistry) Eukaryota Proteins Xanthine superoxide dismutase ANTIOXIDATIVE ENZYMES Cold Temperature NBT/riboflavin assay chemistry Biochemistry marine Antarctic diatom biology.protein GROWTH ULTRAVIOLET-RADIATION |
| Zdroj: | Journal of Photochemistry and Photobiology B: Biology, 87(3), 218-226. ELSEVIER SCIENCE SA Journal of Photochemistry and Photobiology B: Biology, 87(3), 218-226. Elsevier B.V. |
| ISSN: | 1011-1344 |
| Popis: | Antioxidant enzymes such as superoxide dismutase (SOD) play a key role in the removal of reactive oxygen species produced during visible and ultraviolet irradiance stress in microalgae and plants. However, little is known about the enzymatic antioxidative stress responses in ecologically important Antarctic marine microalgae. SOD in particular is difficult to analyze, possibly due to problems in obtaining sufficient quantities necessary for reliable and reproducible enzymatic assays. The aim of the present work was to create a sensitive, easy-to-use and reliable method for SOD determination in Antarctic microalgal material by comparing and optimizing existing protein extraction procedures and SOD assays in the marine Antarctic diatom Chaetoceros brevis. Optimization was achieved in cell disruption (sonication) and protein extraction procedures, extraction buffers, SOD assay methods (xanthine/xanthine oxidase and NBT/ riboflavin photometric quantitative methods and native gel electrophoresis qualitative method) and the assay temperature. Protein extraction was optimal at low sonication amplitudes after a few pulses, irrespective of the type of buffer used. Extraction efficiency varied highly between the tested buffers; most protein was extracted in the presence of 1% of Triton X-100. SOD activity was best quantified using the NBT/riboflavin method in combination with a buffer containing potassium phosphate and Triton X-100. Moreover, the NBT/ riboflavin method was demonstrated to be the most reliable and sensitive method at low temperatures (5 degrees C). (c) 2007 Elsevier B.V. All rights reserved. |
| Databáze: | OpenAIRE |
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