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-oct adecadienoyl)-L-3- phosphatidylcholine (PC-OOH) and 1-palmitoyl-2-(13-hydro xy-cis-9,trans-11-octadecadienoyl)-L-3- phosphatidylcholine (PC-OH) and the quantitative determination of PC-OH, the enzymatic activity of phospholipi d hydroperoxide glutathione peroxidase (PHGPx) can be measured by capillary electrophoresis. The separation was carried out in a fused-silica capillar y (30 cm x 100 mum id) at 15 kV positive voltage. Sodium borate (100 mM; pH = 8.4) was used as the running buffer, and the photodiode array detector w avelength was 232 nm. The determination can be completed in 5 min. The dete ction limit was 5 pmol, and the relative standard deviation (RSD) of the pe ak area was less than 1% with an average recovery of 98.6%. Compared with t raditional methods such as HPLC and spectrophotometry, it is faster and mor e 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) mu mol min(-1), and the specific activity of partially purified trans-gene PHGPx was 3.1 x 10(-2) mu mol min(-1) per mg. The stability of the trans-gene PHGPx was also studied.