Purification and some properties of L-fucose dehydrogenase from Agrobacterium radiobacter and its application to the assay of bound-fucose in glycoconjugates.
L-Fucose dehydrogenase was found in the cell extract of Agrobacterium radiobacter and purified to homogeneity about 480-fold with 16% recovery. The molecular weight of the enzyme was approx. 64,000. The enzyme was active in the neutral pH range, unlike other L-fucose or D-arabinose dehydrogenases which are active only in the alkaline pH range. Using this enzyme and alpha-L-fucosidase F-I of Bacillus circulans (Tsuji, Y., Yamamoto, K., Tochikura, T., Seno, T., Ohkubo, Y. and Yamaguchi, H. (1990) J. Biochem. 107, 324-330) simultaneously, we developed a new coupled enzymatic method in a single buffer system for determining bound-fucose in biological materials. The fucose released by alpha-L-fucosidase F-I was oxidized with L-fucose dehydrogenase in the presence of NAD+, and the NADH formed was measured by absorbance of ultraviolet or utilized to generate color in a reaction involving CuSO4 and neocuproine. Using these methods, bound-fucose in various oligosaccharides and proteins such as lacto-N-fucopentaoses and porcine gastric mucin were quantitated within 15 min.
Tsuji
Y
Y
Research Laboratory of Higashimaru Shoyu Co., Ltd., Hyogo, Japan.
Koike
A
A
Yamamoto
K
K
Tochikura
T
T
D000818:Animals; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D003300:Copper; D019327:Copper Sulfate; D005643:Fucose / Q000032:analysis*; D006001:Glycoconjugates / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009077:Mucins / Q000032:analysis; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010618:Phenanthrolines; D012231:Rhizobium / Q000201:enzymology*; D013053:Spectrophotometry; D013552:Swine; D005644:alpha-L-Fucosidase / Q000378:metabolism
D006001:Glycoconjugates; D009077:Mucins; D010618:Phenanthrolines; D009243:NAD; D005643:Fucose; D003300:Copper; C002701:neocuproine; D002237:Carbohydrate Dehydrogenases; C020154:L-fucose dehydrogenase; D005644:alpha-L-Fucosidase; D019327:Copper Sulfate