A novel fluorescence competitive assay for glucose determinations by using a thermostable glucokinase from the thermophilic microorganism Bacillus stearothermophilus

Sabato D'Auria, Nicolas DiCesare, Maria Staiano, Zygmunt Gryczynski, Mose' Rossi, Joseph R. Lakowicz

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

We describe the use of a thermostable glucokinase in a novel competitive fluorescence assay for glucose. Glucokinase from Bacillus stearothermophilus (BSGK) was found to retain enzymatic activity in solution for over 20 days. The single cysteine residue in BSGK, which is near the active site, was labeled with a fluorescent probe, 2- (4-iodoacetamidoanilino)naphthalene-6-sulfonic acid. The ANS-labeled BSGK displayed a modest 25% decrease in the emission intensity upon binding glucose but no change in lifetime. To obtain a larger spectral change we developed a competitive assay for glucose using the intrinsic tryptophan fluorescence from BSGK and a resonance energy transfer (RET) acceptor-labeled sugar. The sugar-labeled acceptor quenched the BSGK tryptophan emission, and the quenching was reversed upon addition of glucose. The use of RET as the sensing mechanism can be easily extended to longer wavelengths for a more practical glucose sensor.

Original languageEnglish
Pages (from-to)138-144
Number of pages7
JournalAnalytical Biochemistry
Volume303
Issue number2
DOIs
StatePublished - 15 Apr 2002

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Geobacillus stearothermophilus
Glucokinase
Bacilli
Microorganisms
Assays
Fluorescence
Glucose
Tryptophan
Sugars
Energy transfer
Energy Transfer
Glucose sensors
Sulfonic Acids
Fluorescent Dyes
Cysteine
Quenching
Catalytic Domain
Wavelength

Cite this

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title = "A novel fluorescence competitive assay for glucose determinations by using a thermostable glucokinase from the thermophilic microorganism Bacillus stearothermophilus",
abstract = "We describe the use of a thermostable glucokinase in a novel competitive fluorescence assay for glucose. Glucokinase from Bacillus stearothermophilus (BSGK) was found to retain enzymatic activity in solution for over 20 days. The single cysteine residue in BSGK, which is near the active site, was labeled with a fluorescent probe, 2- (4-iodoacetamidoanilino)naphthalene-6-sulfonic acid. The ANS-labeled BSGK displayed a modest 25{\%} decrease in the emission intensity upon binding glucose but no change in lifetime. To obtain a larger spectral change we developed a competitive assay for glucose using the intrinsic tryptophan fluorescence from BSGK and a resonance energy transfer (RET) acceptor-labeled sugar. The sugar-labeled acceptor quenched the BSGK tryptophan emission, and the quenching was reversed upon addition of glucose. The use of RET as the sensing mechanism can be easily extended to longer wavelengths for a more practical glucose sensor.",
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A novel fluorescence competitive assay for glucose determinations by using a thermostable glucokinase from the thermophilic microorganism Bacillus stearothermophilus. / D'Auria, Sabato; DiCesare, Nicolas; Staiano, Maria; Gryczynski, Zygmunt; Rossi, Mose'; Lakowicz, Joseph R.

In: Analytical Biochemistry, Vol. 303, No. 2, 15.04.2002, p. 138-144.

Research output: Contribution to journalArticle

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