Yao K, Ubuka T, Masuoka N, Kinuta M, Ohta J, Teraoka T, Futani S
Department of Biochemistry, Okayama University Medical School, Japan.
J Chromatogr. 1992 Oct 2;581(1):11-5. doi: 10.1016/0378-4347(92)80442-s.
A new assay method for sialidase (EC 3.2.1.18) activity using ion-exchange chromatography and acidic ninhydrin reaction has been developed. Fetuin, 4-methylumbelliferyl-N-acetylneuraminic acid (MUB-NANA), gangliosides and N-acetylneuramin-lactose were examined as substrates. Free sialic acid liberated from these substrates by sialidase reaction was isolated with a Dowex 1-X8 column (trifluoroacetate form, 1.5 cm x 0.5 cm I.D.) and determined by acidic ninhydrin reaction. Among the substrates tested, MUB-NANA was the best in the present method, N-Acetylneuramin-lactose could not be used as the substrate, because it was not separated from liberated sialic acid under the conditions used. The recovery of N-acetylneuraminic acid was above 88%, and the sensitivity of the method was 20 nmol in 300 microliters of the reaction mixture. The method was applied to the sialidase assay during its purification from rat skeletal muscle, and a Michaelis constant of 1.15 mM was obtained with MUB-NANA as the substrate. The method using the acidic ninhydrin reaction was simple and exhibited good reproducibility.
已开发出一种利用离子交换色谱法和酸性茚三酮反应测定唾液酸酶（EC 3.2.1.18）活性的新方法。以胎球蛋白、4-甲基伞形酮基-N-乙酰神经氨酸（MUB-NANA）、神经节苷脂和N-乙酰神经氨酸乳糖作为底物进行检测。通过唾液酸酶反应从这些底物中释放出的游离唾液酸，用Dowex 1-X8柱（三氟乙酸形式，内径1.5 cm×0.5 cm）进行分离，并通过酸性茚三酮反应进行测定。在所测试的底物中，MUB-NANA在本方法中表现最佳，N-乙酰神经氨酸乳糖不能用作底物，因为在所用条件下它无法与释放出的唾液酸分离。N-乙酰神经氨酸的回收率高于88%，该方法的灵敏度为300微升反应混合物中20纳摩尔。该方法应用于从大鼠骨骼肌中纯化唾液酸酶过程中的活性测定，以MUB-NANA为底物时得到的米氏常数为1.15 mM。使用酸性茚三酮反应的方法简单且重现性良好。
