Fluorescence assay of glucosylceramide glucosidase using NBD-cerebroside.
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.
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Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor.
Shayman
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Radin
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D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D002518:Ceramides / Q000032:analysis*; D002554:Cerebrosides / Q000378:metabolism*; D000431:Ethanol / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D005470:Fluorometry / Q000379:methods* / Q000592:standards; D005962:Glucosylceramidase / Q000032:analysis*
D002518:Ceramides; D002554:Cerebrosides; D005456:Fluorescent Dyes; C079364:NBD-cerebroside; D000431:Ethanol; C037974:N-(7-(4-nitrobenzo-2-oxa-1,3-diazole))-6-aminocaproyl sphingosine; D005962:Glucosylceramidase; D009327:4-Chloro-7-nitrobenzofurazan