Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho Naka Sakai, Osaka, 599-8531, Japan.
Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and Technology (PRESTO), 7 Goban-cho Chiyoda, Tokyo, 102-0076, Japan.
Anal Sci. 2021 Oct 10;37(10):1439-1446. doi: 10.2116/analsci.21P067. Epub 2021 Apr 9.
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.
开发了一种使用堵塞水凝胶的新型电动过滤装置，用于直接测量酶反应的初始速率。在提出的方法中，当底物通过被水凝胶捕获的酶的薄层时，酶反应仅在很短的时间内进行，而没有混合和检测的滞后时间。在实验条件下，通过分子筛效应将碱性磷酸酶（酶）过滤到堵塞水凝胶的阴极侧界面，提供了厚度约为 100μm 的薄酶区。当将酶捕获后，将 4-甲基伞形酮磷酸盐（底物）通过电泳引入到装置中时，由于底物穿过捕获的酶区，仅在 1.26s 内就产生了 4-甲基伞形酮（产物）。因此，通过简单地将产物的浓度除以可调的反应时间，可直接将酶反应的初始速率计算为 31.0μM/s。与混合酶和底物溶液获得的初始速率相比，由于通过捕获浓缩了酶，酶反应的最大速度值是混合方法的 30 倍。在该方法中，米氏常数是混合方法的 2.7 倍，表明在实验条件下，形成复合物的平衡变化。
