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Fluorescence assay of glucosylceramide glucosidase using NBD‐Cerebroside
Author(s) -
Abe Akira,
Shayman James A.,
Radin Norman S.
Publication year - 1992
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/bf02535587
Subject(s) - chemistry , fluorescence , cerebroside , yield (engineering) , substrate (aquarium) , chromatography , hydrolysis , ceramide , moiety , ethanol , methylparaben , biochemistry , photochemistry , organic chemistry , biology , apoptosis , physics , materials science , quantum mechanics , metallurgy , ecology , preservative
A sensitive fluorometric assay for glucocerebroside β‐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, Slow Fade. 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.