Monitoring the activity of ribonucleotide reductase by capillary zone electrophoresis

• Main Authors: Yen-Ching Liu, 劉儼慶
• Other Authors: Huey-Fen Tzeng
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/96961971112659001007

碩士 === 國立暨南國際大學 === 應用化學系 === 98 === Ribonucleotide reductase (RR) is an important enzyme for DNA biosynthesis. Its activity is tremendously dependent on cell cycle and cell proliferation. In this study, we developed a capillary zone electrophoretic method with a reversed electroosmotic flow to determine 2’-deoxycytidine5’-diphosphate (dCDP) from the reduction of cytidine 5’-diphosphate (CDP) for RR assays. In order to increase the mobility difference of dCDP and CDP, we added hydroxypropyl-α-cyclodextrin (HP-α-CD) and phenylboronic acid into the background electrolyte (BGE). The hydrophobic interaction between HP-α-CD and the phenyl group of phenylboronic acid as well as the condensation of borate with ribonucleotide makes the mobility of CDP-phenylboronic acid-HP-α-CD complex decreased, so we can directly analyze RR assay mixture. We also used a cationic polymer, polyethylenimine, to dynamically coat the inner surface of the uncoated capillary, which made electroosmotic flow reversed. Therefore electrokinetic injection, could introduce much more sample into capillary than pressure injection. Because the viscosity and conductivity difference between the sample zone and BGE zone, when analytes move into BGE, their velocities decrease, which results in online preconcentration and the improvement of the detection limit. Herein, we investigated the effects of phenylboronic acid, HP-α-CD, ethylenediaminetetraacetic acid, polyethylenimine and BGE. Under optimal conditions, it takes 10 min to finish the analysis. The linear range of this method was 0.5–100 μM for dCDP and dCTP, and the correlation coefficients were 0.999 and 0.998 for dCDP and dCTP, respectively. The detection limits of dCDP and dCTP were 1.2 μM and 0.75 μM for dCDP and dCTP, respectively.