SPECTROPHOTOMETRIC DETERMINATION OF L-ASPARAGINE BY FLOW-INJECTION ANALYSIS USING L-ASPARAGINASE IMMOBILIZED ON AN EPOXY-RESIN

A continuous now procedure for the determination of L-asparagine based on its L-asparaginase-catalyzed conversion to L-aspartate and ammonia is proposed. The ammonia formed is measured via the indophenol blue generated in Berthelot's reaction. The enzyme L-asparaginase is covalently bound to an epoxy resin (VA-Epoxy Biosynth, Riedel-De Haen) packed in a glass column(50 X 3 mm i.d.). This enzyme reactor can be used for more than 5 months and maintains about 88% of its initial enzyme activity after 700 determinations. Under the proposed experimental conditions a linear calibration range is obtained for L-asparagine concentrations between 60 mu M and 15 mM (Abs = -4.29 X 10(-3) + 8.05 X 10(-4)[Asp]; r = 0.999). The proposed procedure has a detection limit (3 sigma) of 1.2 ppm (9 mu M) and a relative standard deviation of 1.9% (12 determinations at 30 ppm). Among the 22 amino acids investigated, only L-histidine (greater than or equal to 960 ppm) and L-methionine (greater than or equal to 640 ppm) were found to interfere.