Colorimetric assay for free and bound L-fucose.
A novel, rapid, and reliable colorimetric method for measuring L-fucose has been developed. This method utilizes NADH formed from the interaction of L-fucose with fucose dehydrogenase and NAD to generate color in a reaction involving CuSO4 and neocuproine. NADH reduces Cu2+ to Cu1+ and the latter interacts with neocuproine to yield a complex with a maximal absorption at 455 nm. The reaction of NADH with copper-neocuproine is immediate and under the conditions of the assay the color formed remains stable for at least 2 h. When the assay is used to determine levels of L-fucose, the absorbance is found to be linearly proportional to exogenously added fucose concentrations from 16 to 179 nmol with resulting molar extinction coefficient of 13,660. Using this procedure, L-fucose released by acid hydrolysis from porcine submaxillary mucin, and by alpha-L-fucosidase from p-nitrophenyl-alpha-L-fucopyranoside, was quantitated.
Cohenford
M A
MA
Scott Laboratories, Inc., West Warwick, Rhode Island 02893.
Abraham
A
A
Abraham
J
J
Dain
J A
JA
D000818:Animals; D002237:Carbohydrate Dehydrogenases / Q000378:metabolism; D002854:Chromatography, Paper; D003300:Copper; D005643:Fucose / Q000032:analysis*; D006027:Glycosides; D006868:Hydrolysis; D009077:Mucins; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010618:Phenanthrolines; D013053:Spectrophotometry; D013552:Swine; D005644:alpha-L-Fucosidase / Q000378:metabolism
D006027:Glycosides; D009077:Mucins; D010618:Phenanthrolines; D009243:NAD; C014125:4-nitrophenylfucoside; D005643:Fucose; D003300:Copper; C002701:neocuproine; D002237:Carbohydrate Dehydrogenases; C020154:L-fucose dehydrogenase; D005644:alpha-L-Fucosidase