Alon R, Bayer E A, Wilchek M
Department of Biophysics, Weizmann Institute of Science, Rehovot, Israel.
J Biochem Biophys Methods. 1991 Jan;22(1):23-33. doi: 10.1016/0165-022x(91)90078-b.
A multi-coupled enzyme assay system for determining sialidase activity is described. Enzymes, substrates and chromogens are reacted in situ and determined spectrophotometrically in ELISA microtiter plates. Sialidase is assayed by the extent of desialylated galactose on an appropriate sialoglycoconjugate (fetuin), which is otherwise unavailable for oxidation by galactose oxidase. The oxidation is monitored by the coupling of H2O2 released to a third enzyme, peroxidase. The rate of change of absorbance at 405 nm, resulting from the oxidized chromogen is a measure of the reaction rate of the coupled enzyme system. A similar system can be used for determining galactose oxidase in solution, or on blots using galactose as substrate. Due to the small-scale single-step measurement, the described assay is a sensitive, convenient, and inexpensive alternative to the classic colorimetric determination.
描述了一种用于测定唾液酸酶活性的多耦合酶分析系统。酶、底物和显色剂在酶联免疫吸附测定（ELISA）微孔板中进行原位反应，并通过分光光度法进行测定。唾液酸酶通过适当的唾液糖缀合物（胎球蛋白）上脱唾液酸化半乳糖的程度来测定，否则该半乳糖不能被半乳糖氧化酶氧化。通过将释放的过氧化氢与第三种酶（过氧化物酶）偶联来监测氧化反应。由氧化显色剂导致的405nm处吸光度的变化速率是耦合酶系统反应速率的一种度量。类似的系统可用于测定溶液中的半乳糖氧化酶，或在使用半乳糖作为底物的印迹上进行测定。由于是小规模单步测量，所描述的分析方法是一种比经典比色法更灵敏、方便且廉价的替代方法。
