Assay of sialidase activity using ion-exchange chromatography and acidic ninhydrin reaction

Kenzaburoh Yao, Toshihiko Ubuka, Noriyoshi Masuoka, Masahiro Kinuta, Jun Ohta, Toshito Teraoka, Shinya Futani

Research output: Contribution to journalArticle

5 Citations (Scopus)

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 × 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 μl 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.

Original languageEnglish
Pages (from-to)11-15
Number of pages5
JournalJournal of Chromatography B: Biomedical Sciences and Applications
Volume581
Issue number1
DOIs
Publication statusPublished - Oct 2 1992

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Ninhydrin
Ion Exchange Chromatography
Neuraminidase
N-Acetylneuraminic Acid
Chromatography
Assays
Ion exchange
Substrates
Fetuins
Trifluoroacetic Acid
Gangliosides
Purification
Muscle
Rats
Skeletal Muscle
Recovery

ASJC Scopus subject areas

  • Chemistry(all)
  • Clinical Biochemistry
  • Molecular Medicine

Cite this

Assay of sialidase activity using ion-exchange chromatography and acidic ninhydrin reaction. / Yao, Kenzaburoh; Ubuka, Toshihiko; Masuoka, Noriyoshi; Kinuta, Masahiro; Ohta, Jun; Teraoka, Toshito; Futani, Shinya.

In: Journal of Chromatography B: Biomedical Sciences and Applications, Vol. 581, No. 1, 02.10.1992, p. 11-15.

Research output: Contribution to journalArticle

Yao, Kenzaburoh ; Ubuka, Toshihiko ; Masuoka, Noriyoshi ; Kinuta, Masahiro ; Ohta, Jun ; Teraoka, Toshito ; Futani, Shinya. / Assay of sialidase activity using ion-exchange chromatography and acidic ninhydrin reaction. In: Journal of Chromatography B: Biomedical Sciences and Applications. 1992 ; Vol. 581, No. 1. pp. 11-15.
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