Capillary Electrophoresis/Dynamic Frontal Analysis for the Enzyme Assay of 4-Nitrophenyl Phosphate with Alkaline Phosphatase.

A substrate of 4-nitrophenyl phosphate was enzymatically hydrolyzed by alkaline phosphatase (ALP) in a capillary tube, while an injected zone of the substrate was electrophoretically migrating in the separation buffer containing the enzyme by capillary electrophoresis (CE). During CE migration of the substrate from the start time of the electrophoresis to the detection time of the substrate, the substrate was continuously hydrolyzed by ALP to form a product of 4-nitrophenolate, and a plateau signal of 4-nitrophenolate was detected as a result of the zero-order kinetic reaction. The height of the plateau signal was directly related to the reaction rate, and it was used for the determination of a Michaelis-Menten constant through Lineweaver-Burk plots. Since the plateau signal is attributed to the dynamic formation of the product by the enzymatic reaction in CE, this analysis method is named as capillary electrophoresis/dynamic frontal analysis (CE/DFA). In CE/DFA, the CE separation is included on detecting the plateau signal, and the hydrolysis product before the sample injection is resolved from the dynamically and continuously formed product. The inhibition of the enzyme with the product is also eliminated in CE/DFA by the CE separation.