A highly sensitive capillary electrophoresis method using p-nitrophenyl 5 %26apos;-thymidine monophosphate as a substrate for the monitoring of nucleotide pyrophosphatase/phosphodiesterase activities

作者：Lee Sang Yong; Levesque Sebastien A; Sevigny Jean; Mueller Christa E

来源：Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2012, 911: 162-169.

DOI：10.1016/j.jchromb.2012.10.044

• AOPCP
• Benzoylbenzoyl-ATP
• Capillary electrophoresis
• Dial-ADP
• p-Nitrophenyl 5 %26apos
• -thymidine monophosphate
• NPP inhibitors
• NPP substrates
• Nucleotide pyrophosphatase
• Polybrene

A highly sensitive capillary electrophoresis method has been developed to monitor the activity of nucleotide pyrophosphatases/phosphodiesterases (NPPs) and screen for NPP inhibitors. In this method, p-nitrophenyl 5%26apos;-thymidine monophosphate (p-Nph-5%26apos;-TMP) was used as an artificial substrate, and separation of reaction products was performed on a dynamically coated capillary. We found that the optimal capillary electrophoresis (CE) conditions were as follows: fused-silica capillary (20 cm effective length x 75.5 mu m (id)), electrokinetic injection for 60s, 70 mM phosphate buffer containing polybrene 0.002%, pH 9.2, constant current of -80 mu A, constant capillary temperature of 15 degrees C and detection at 400 nm. To allow precise quantification, 2-methyl-4,6-dinitrophenol (dinitrocresol) was applied as an internal standard. The limit of detection (LOD) and the limit of quantification (LOQ) were 137 and 415 nM, respectively. This new method was shown to be over 8-fold more sensitive than the conventional spectrophotometric assays and 16-fold more than the previously reported CE procedure, and the results (K-m values for NPP1 and NPP3, K-i values for standard inhibitors) obtained were in accordance with previous literature data. Therefore, this new method is an improvement of actual techniques and could be used as a quick and standard analytical technique for the identification and characterization of NPP inhibitors.

• 出版日期2012-12-12