Enzymatic activity measurement of phospholipid hydroperoxide glutathione peroxidase by capillary electrophoresis.
Based on the separation of 1-palmitoyl-2-(13-hydroperoxy-cis-9,trans-11-octadecadienoyl)-L-3- phosphatidylcholine (PC-OOH) and 1-palmitoyl-2-(13-hydroxy-cis-9,trans-11-octadecadienoyl)-L-3- phosphatidylcholine (PC-OH) and the quantitative determination of PC-OH, the enzymatic activity of phospholipid hydroperoxide glutathione peroxidase (PHGPx) can be measured by capillary electrophoresis. The separation was carried out in a fused-silica capillary (30 cm x 100 microns id) at 15 kV positive voltage. Sodium borate (100 mM; pH = 8.4) was used as the running buffer, and the photodiode array detector wavelength was 232 nm. The determination can be completed in 5 min. The detection limit was 5 pmol; and the relative standard deviation (RSD) of the peak area was less than 1% with an average recovery of 98.6%. Compared with traditional methods such as HPLC and spectrophotometry, it is faster and more convenient. Using capillary electrophoresis, the enzymatic activities of PHGPx expressed by the rice PHGPx gene in E. coli. M15 was determined as 1.25 x 10(-5) mumol min-1, and the specific activity of partially purified trans-gene PHGPx was 3.1 x 10(-2) mumol min-1 per mg. The stability of the trans-gene PHGPx was also studied.
Ru
Q H
QH
School of Life Science and Engineering, Tsinghua University, Beijing, 100084, China.
Luo
G A
GA
Wang
Z B
ZB
Liu
J Y
JY
D002138:Calibration; D019075:Electrophoresis, Capillary; D004926:Escherichia coli / Q000201:enzymology; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism*; D012275:Oryza / Q000235:genetics; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D019076:Transgenes
D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase