Ethanol unfolds firefly luciferase while competitive inhibitors antagonize unfolding: DSC and FTIR analyses

https://doi.org/10.1016/0731-7085(94)00045-XGet rights and content

Abstract

Firefly luciferase has gained popularity as a protein model in elucidating anaesthesia mechanism because the bioluminescence of the purified enzyme system is extremely sensitive to volatile anaesthetics. This study analysed the thermal unfolding of firefly luciferase by differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR). DSC showed that the transition of firefly luciferase from the folded (N) to unfolded (D) state occurred at 41.7°C with the excess heat flow of 1.6 cal g−1 protein. Ethanol decreased the transition temperature dose dependently. In contrast, luciferin competitors, anilinonaphthalenesulphonate (ANS), toluidinonaphthalenesulphonate (TNS), and myristic acid increased the transition temperature. The competitive inhibitors antagonized unfolding and stabilized the N-state. Ethanol promoted unfolding and stabilized the D-state. Temperature scan by FTIR agreed with the DSC data. The intensities of amide-I′ and amide-II′ bands started to increase at 20–25°C. This temperature coincides with the temperature where the bioluminescence of firefly luciferase is maximal. The unfolding effect of ethanol was evident even at 5°C. ANS, TNS, and myristic acid completely protected the enzyme from the thermal unfolding. This is the first demonstration that the noncompetitive inhibitors induce the isothermal first-order phase transition in a functional protein, whereas competitive inhibitors protect the enzyme from thermal unfolding. The action mode of competitive inhibitors on firefly luciferase is completely different from that of noncompetitive inhibitors.

References (30)

  • A.A. Green et al.

    Biochim. Biophys. Acta

    (1956)
  • A. Shibata et al.

    J. Pharm. Sci.

    (1991)
  • J.S. Chiou et al.

    Biochim. Biophys. Acta

    (1992)
  • P.D. Boyer et al.

    J. Biol. Chem.

    (1946)
  • J.S. Chiou et al.

    Biochim. Biophys. Acta

    (1992)
  • M. Jackson et al.

    Biochim. Biophys. Acta

    (1989)
  • I. Ueda

    Anesthesiology

    (1965)
  • W.D. McElroy et al.

    Fed. Proc.

    (1962)
  • I. Ueda et al.

    Anesthesiology

    (1973)
  • N.P. Franks et al.

    Nature

    (1984)
  • N.P. Franks et al.

    Nature

    (1985)
  • S. Ainsworth
    (1977)
  • M. DeLuca

    Biochemistry

    (1969)
  • A. Shibata et al.

    Biochemistry

    (1992)
  • J. Brandt et al.

    Int. Peptide Protein Res.

    (1976)
  • Cited by (21)

    • Ethanol's Action Mechanisms in the Brain: From Lipid General Alterations to Specific Protein Receptor Binding

      2017, Addictive Substances and Neurological Disease: Alcohol, Tobacco, Caffeine, and Drugs of Abuse in Everyday Lifestyles
    • Separation and detection techniques for peptides and proteins in stability research and bioanalysis

      2000, Journal of Chromatography B: Biomedical Sciences and Applications
    • Halothane, an inhalational anesthetic agent, increases folding stability of serum albumin

      1999, Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
    View all citing articles on Scopus

    Presented at the Eighth Annual American Association of Pharmaceutical Scientists Meeting, November 1993, Orlando, Florida, USA.

    View full text