Michaelis-Menten Analysis of Alkaline Phosphatase by Capillary Electrophoresis Using Plug-Plug Reaction

Abstract

This work evaluated the use of an alkaline phosphatase plug, slowly migrating in a polyacrylamide-coated capillary filled with buffer/soluble polymer, to convert p-nitrophenylphosphate (which was injected into the capillary as a separate plug, and migrated faster than the enzyme) to p-nitrophenol under a constant applied potential. The elution of assay components was monitored on-capillary at 230 nm. The initial reaction velocity of the enzymecatalyzed reaction was estimated from the peak area ratio of the enzyme product to the internal standard. Using the Lineweaver-Burk plots, an average Michaelis constant (K_M) for alkaline phosphatase was calculated to be 4.8 ± 0.3 mM (n = 4, CV = 6%). With the constant potential electrophoresis (8 kV/57 cm), the method had a detection limit of 4.4 × 10⁻⁵ IU for alkaline phosphatase and a linear range up to 2.1 × 10⁻³ IU.