title: Assay of sialidase activity using ion-exchange chromatography and acidic ninhydrin reaction.
abstract: A new assay method for sialidase (EC 3.2.1.18) activity using ion-exchange chromatography and acidic ninhydrin reaction has been developed. Fetuin, 4-methylumbelliferyl-N-acetylneuraminic acid (MUB-NANA), gangliosides and N-acetylneuramin-lactose were examined as substrates. Free sialic acid liberated from these substrates by sialidase reaction was isolated with a Dowex 1-X8 column (trifluoroacetate form, 1.5 cm x 0.5 cm I.D.) and determined by acidic ninhydrin reaction. Among the substrates tested, MUB-NANA was the best in the present method, N-Acetylneuramin-lactose could not be used as the substrate, because it was not separated from liberated sialic acid under the conditions used. The recovery of N-acetylneuraminic acid was above 88%, and the sensitivity of the method was 20 nmol in 300 microliters of the reaction mixture. The method was applied to the sialidase assay during its purification from rat skeletal muscle, and a Michaelis constant of 1.15 mM was obtained with MUB-NANA as the substrate. The method using the acidic ninhydrin reaction was simple and exhibited good reproducibility.
authors.affiliation: Department of Biochemistry, Okayama University Medical School, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Yao
authors.forename: T
authors.initials: T
authors.lastname: Ubuka
authors.forename: N
authors.initials: N
authors.lastname: Masuoka
authors.forename: M
authors.initials: M
authors.lastname: Kinuta
authors.forename: J
authors.initials: J
authors.lastname: Ohta
authors.forename: T
authors.initials: T
authors.lastname: Teraoka
authors.forename: S
authors.initials: S
authors.lastname: Futani
mesh_terms: D000818:Animals; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008297:Male; D009132:Muscles / Q000201:enzymology; D009439:Neuraminidase / Q000032:analysis*; D009555:Ninhydrin / Q000737:chemistry*; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity
chemical_list: D007202:Indicators and Reagents; D009439:Neuraminidase; D009555:Ninhydrin