TY - JOUR
T1 - A comparison of quantitative and qualitative superoxide dismutase assays for application to low temperature microalgae
AU - Janknegt, P.J.
AU - Rijstenbil, J.W.
AU - van de Poll, W.H.
AU - Gechev, T.S.
AU - Buma, A.G.J.
N1 - Reporting year: 2007
Metis note: 4156;CEME; MM; file:///C:/pdfs/Pdfs2007/Janknegt_ea_4156.pdf
PY - 2007
Y1 - 2007
N2 - 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 °C).
AB - 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 °C).
U2 - 10.1016/j.jphotobiol.2007.04.002
DO - 10.1016/j.jphotobiol.2007.04.002
M3 - Article
SN - 1011-1344
VL - 87
SP - 218
EP - 226
JO - Journal of Photochemistry and Photobiology B: Biology
JF - Journal of Photochemistry and Photobiology B: Biology
IS - 3
ER -