Catalytic Spectrophotometric Quantitation for Hypoxanthine by Conjugating Xanthine Oxidase with Horseradish Peroxidase
LI Zhong-qin1,2,XU Xiao-ping1,2*,WANG Wu1
1. The Key Laboratory of Industry Biotechnology, Ministry of Education, Southern Yangtze University, Wuxi 214036, China 2. College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 350002, China
Abstract：A novel technology to determine the concentration of hypoxanthine through the chromogenic reaction of phenic acid (PA), 4-aminoantipyrine(AAP) and hydrogen peroxide (H2O2), which was produced via the oxidation of hypoxanthine catalyzed by xanthine oxidase(XO), under the help of horseradish peroxidase(HRP) was proposed in the present paper, according to the reaction of molybdoenzyme xanthine oxidase (XOD, EC 1.2.3.22) which mainly catalyzes the conversion of hypoxanthine and xanthine to xanthine and uric acid, respectively, and is capable of reducing oxygen to generate the reactive oxygen species (ROS), superoxide and hydrogen peroxide. The influences of temperature and pH on the system were investigated. The optimal conditions to determine the concentration of hypoxanthine were obtained as follows: XO(0.32 U·mL-1), HRP(7.0U·mL-1), 4-Aminoantipyrine(1 mmol·L-1), and phenic acid(6 mmol·L-1) were dissolved in 100 mmol·L-1 Tris-HCl buffer solution (pH 8.3), and the reaction system was incubated in thermostat of 37 ℃ for 8 min, the absorptive wave length was 508 nm. Under the conditions mentioned above, the linear range of calibration curve was between 0.2 and 3.0 mmol·L-1, the correlation coefficient was 0.997 9, and the limit of detection was 0.05 mmol·L-1. All these show that this technology is a potential alternative method to determine the concentration of hypoxanthine in areas like for example in laboratory or clinical serum diagnosis.
