A novel electrokinetic filtration device using a plugged hydrogel was developed to directly measure the initial rate of enzyme reactions. In the proposed method, the enzyme reaction proceeded only for a short time when the substrate was passed through a thin layer of enzyme trapped by the hydrogel without any lag times for mixing and detection. In experimental conditions, alkaline phosphatase (enzyme) was filtrated at a cathodic-side interface of the plugged hydrogel by molecular sieving effect, providing the thin enzyme zone whose thickness was approximately 100 μm. When 4-methylumberiferyl phosphate (substrate) was electrokinetically introduced into the device after trapping the enzyme, 4-methylumberiferone (product) was generated by the enzyme reaction for only 1.26 s as the substrate passed through the trapped enzyme zone. As a result, the initial rate of the enzyme reaction could be directly calculated to 31.0 μM/s by simply dividing the concentration of the product by the tunable reaction time. Compared to the initial rate obtained by mixing the enzyme and substrate solutions, the value of the maximum velocity of the enzyme reaction was 30-fold larger than that in the mixing method due to the preconcentration of the enzyme by trapping. The Michaelis-Menten constant in the proposed method was 2.7-fold larger than that in the mixing method, suggesting the variation of changes in the equilibrium of complex formation under the experimental conditions.
Keywords: Enzyme; electrokinetic filtration; gel electrophoresis; initial rate of enzyme reaction.