Abe A, Shayman J A, Radin N S
Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor.
Lipids. 1992 Dec;27(12):1052-4. doi: 10.1007/BF02535587.
A sensitive fluorometric assay for glucocerebroside beta-glucosidase [Dinur, T., Grabowski, G.A., Desnick, R.J., and Gatt, S. (1984) Anal. Biochem. 136, 223-234] has been reexamined. It was found that the lipids containing the NBD moiety (12-[N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)] used for standardization of the assay are light-sensitive and that the yield of fluorescent light is very sensitive to the composition of the solvent used in the fluorometric measurement. Some protection against fading could be obtained by adding a free-radical trapping agent, SlowFade. The fading of the free NBD-acid, when used for standardization, could be prevented by adding ethanol to the solvent, but this reduced the fluorescence yield. It is recommended that some of the fluorescent substrate be enzymatically hydrolyzed completely to NBD-ceramide, which can be utilized as the standard without the need to add ethanol. A warning about enzyme reaction rate stability with time is given, with a suggestion for ensuring constancy of activity.
已对一种用于检测葡糖脑苷脂β-葡萄糖苷酶的灵敏荧光测定法[迪努尔，T.，格拉博夫斯基，G.A.，德斯尼克，R.J.，以及加特，S.（1984年）《分析生物化学》136卷，223 - 234页]进行了重新审视。结果发现，用于该测定法标准化的含有NBD部分（12 - [N - 甲基 - N - （7 - 硝基苯并 - 2 - 恶唑 - 1,3 - 二氮杂环丁烷 - 4 - 基）]）的脂质对光敏感，并且荧光产量对荧光测量中所用溶剂的组成非常敏感。通过添加自由基捕获剂SlowFade可以获得一定程度的防褪色效果。用于标准化的游离NBD - 酸的褪色可通过向溶剂中添加乙醇来防止，但这会降低荧光产量。建议将部分荧光底物通过酶促反应完全水解为NBD - 神经酰胺，其可作为标准品使用而无需添加乙醇。给出了关于酶反应速率随时间稳定性的警告，并提出了确保活性恒定的建议。
