Enzymatic activity measurement of phospholipid hydroperoxide glutathione peroxidase by capillary electrophoresis

• Published in: Analyst (London) Vol. 125; no. 11; pp. 1924 - 1927
• Main Authors: RU, Qin-Hua, LUO, Guo-An, WANG, Zhe-Bin, LIU, Jin-Yuan
• Format: Journal Article
• Language: English
• Published: Cambridge Royal Society of Chemistry 01.01.2000
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Calibration; Electrophoresis, Capillary; Enzymes and enzyme inhibitors; Escherichia coli - enzymology; Fundamental and applied biological sciences. Psychology; Glutathione Peroxidase - genetics; Glutathione Peroxidase - metabolism; Oryza - genetics; Oxidoreductases; Transgenes; Monocotyledones; Enzyme; Capillary electrophoresis; Phospholipid-hydroperoxide glutathione peroxidase; Oryza sativa; Peroxidases; Enzymatic activity; Gramineae; Angiospermae; Spermatophyta; Oxidoreductases; Measurement method; Recombinant protein
• ISSN: 0003-2654; 1364-5528
• DOI: 10.1039/b003947k

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.