Denaturation of the Bacillus stearothermophilus dihydrolipoamide dehydrogenase in the presence of guanidine-HCL at low temperature

Yasuaki Hiromasa, Kohji Meno, Yoichi Aso

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Abstract

Denaturation of the Bacillus stearothermophilus dihydrolipoamide dehydrogenase induced by incubation with guanidine-HCl (GdnHCl) at 4°C was examined. Enzyme activity was amplified by 2.5 times in 0.2 M GdnHCl; such an increase in activity was also detected in NaCl and KCl at concentrations less than 1 M, but not in urea. With increasing amount of GdnHCl, the enzyme lost half and all of its activity in 1.0 M and 1.6 M GdnHCl, respectively. Notable changes in fluorescence spectra of Trp residue and FAD cofactor besides circular dichroism in far UV region were detected and insufficiently correlated to the inactivation. Based on changes in fluorescence intensities and molecular ellipticity, a two-state transition equilibrium was suggested; transition midpoints were roughly at 1.7 M GdnHCl. In GdnHCl at concentrations above 2 M, the enzyme released FAD and formed inactive aggregate. Effects by GdnHCl at concentrations less than 1.4 M were mostly cancelled by its removal. Most results were similar to those from studies on the enzyme being one of components of pyruvate dehydrogenase complex.

Original languageEnglish
Pages (from-to)387-394
Number of pages8
JournalJournal of the Faculty of Agriculture, Kyushu University
Volume47
Issue number2
Publication statusPublished - Feb 1 2003

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Dihydrolipoamide Dehydrogenase
Geobacillus stearothermophilus
guanidines
Guanidine
denaturation
Temperature
temperature
Flavin-Adenine Dinucleotide
Enzymes
pyruvate dehydrogenase (lipoamide)
enzymes
Fluorescence
fluorescence
Pyruvate Dehydrogenase Complex
circular dichroism spectroscopy
Circular Dichroism
Urea
inactivation
urea
enzyme activity

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Agronomy and Crop Science

Cite this

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title = "Denaturation of the Bacillus stearothermophilus dihydrolipoamide dehydrogenase in the presence of guanidine-HCL at low temperature",
abstract = "Denaturation of the Bacillus stearothermophilus dihydrolipoamide dehydrogenase induced by incubation with guanidine-HCl (GdnHCl) at 4°C was examined. Enzyme activity was amplified by 2.5 times in 0.2 M GdnHCl; such an increase in activity was also detected in NaCl and KCl at concentrations less than 1 M, but not in urea. With increasing amount of GdnHCl, the enzyme lost half and all of its activity in 1.0 M and 1.6 M GdnHCl, respectively. Notable changes in fluorescence spectra of Trp residue and FAD cofactor besides circular dichroism in far UV region were detected and insufficiently correlated to the inactivation. Based on changes in fluorescence intensities and molecular ellipticity, a two-state transition equilibrium was suggested; transition midpoints were roughly at 1.7 M GdnHCl. In GdnHCl at concentrations above 2 M, the enzyme released FAD and formed inactive aggregate. Effects by GdnHCl at concentrations less than 1.4 M were mostly cancelled by its removal. Most results were similar to those from studies on the enzyme being one of components of pyruvate dehydrogenase complex.",
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AU - Aso, Yoichi

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N2 - Denaturation of the Bacillus stearothermophilus dihydrolipoamide dehydrogenase induced by incubation with guanidine-HCl (GdnHCl) at 4°C was examined. Enzyme activity was amplified by 2.5 times in 0.2 M GdnHCl; such an increase in activity was also detected in NaCl and KCl at concentrations less than 1 M, but not in urea. With increasing amount of GdnHCl, the enzyme lost half and all of its activity in 1.0 M and 1.6 M GdnHCl, respectively. Notable changes in fluorescence spectra of Trp residue and FAD cofactor besides circular dichroism in far UV region were detected and insufficiently correlated to the inactivation. Based on changes in fluorescence intensities and molecular ellipticity, a two-state transition equilibrium was suggested; transition midpoints were roughly at 1.7 M GdnHCl. In GdnHCl at concentrations above 2 M, the enzyme released FAD and formed inactive aggregate. Effects by GdnHCl at concentrations less than 1.4 M were mostly cancelled by its removal. Most results were similar to those from studies on the enzyme being one of components of pyruvate dehydrogenase complex.

AB - Denaturation of the Bacillus stearothermophilus dihydrolipoamide dehydrogenase induced by incubation with guanidine-HCl (GdnHCl) at 4°C was examined. Enzyme activity was amplified by 2.5 times in 0.2 M GdnHCl; such an increase in activity was also detected in NaCl and KCl at concentrations less than 1 M, but not in urea. With increasing amount of GdnHCl, the enzyme lost half and all of its activity in 1.0 M and 1.6 M GdnHCl, respectively. Notable changes in fluorescence spectra of Trp residue and FAD cofactor besides circular dichroism in far UV region were detected and insufficiently correlated to the inactivation. Based on changes in fluorescence intensities and molecular ellipticity, a two-state transition equilibrium was suggested; transition midpoints were roughly at 1.7 M GdnHCl. In GdnHCl at concentrations above 2 M, the enzyme released FAD and formed inactive aggregate. Effects by GdnHCl at concentrations less than 1.4 M were mostly cancelled by its removal. Most results were similar to those from studies on the enzyme being one of components of pyruvate dehydrogenase complex.

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