A direct continuous spectrophotometric assay for glycosidases with 3-nitro-2-pyridyl glycosides by tautomerization of 2-hydroxy-3-nitropyridine

Seung Jin Ma, Jun Hiratake, Yasushi Nakai, Minoru Izumi, Koichi Fukase, Shoichi Kusumoto, Kanzo Sakata

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Two kinds of 3-nitro-2-pyridyl glycosides were synthesized and evaluated as substrates for continuous spectrophotometric assay for glycosidases. The liberated aglycon, 2-hydroxy-3-nitropyridine, immediately tautomerized to 3-nitro-2(1H)-pyridone, causing an absorption shift of ca. 60 nm even under acidic conditions (pH 3-6). Consequently, the enzymatic hydrolysis of these glycosides was monitored continuously in the acidic to neutral pH range (pH 4-7), the optimum pH for most glycosidases. The absorbance of liberated aglycon increased linearly at 390 nm until 10% consumption of the substrate to enable the initial rate to be determined at once without terminating the reaction. The kinetic parameters for the hydrolysis of 3-nitro-2-pyridyl glycosides were obtained from the slopes of the progress curves and were compared with those obtained from the conventional discontinuous assay using p- and o-nitrophenyl glycosides as substrates. The kinetic parameters indicated that 3-nitro-2-pyridyl glycosides were more activated and specific substrates, but with less affinity to the enzymes than the corresponding nitrophenyl glycosides. Moreover, the absorbance shift by tautomerization should promise further applications to continuous spectrophotometric assays for other enzymes acting under acidic conditions, such as acid proteases and acid phosphatases.

Original languageEnglish
Pages (from-to)291-297
Number of pages7
JournalAnalytical Biochemistry
Volume302
Issue number2
DOIs
Publication statusPublished - Jan 1 2002
Externally publishedYes

Keywords

  • 2-hydroxy-3-nitropyridine
  • 3-nitro-2(1H)- pyridone
  • 3-nitro-2-pyridyl glycoside
  • Continuous spectrophotometric assay
  • Glycosidase
  • Tautomerization

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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