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Halothane binding to soluble proteins determined by photoaffinity labeling

R G Eckenhoff1, H Shuman

  • 1Department of Anesthesia, University of Pennsylvania Medical Center, Philadelphia 19104.

Anesthesiology
|July 1, 1993
PubMed
Summary

Photoaffinity labeling reveals halothane binds saturably to proteins like serum albumin, suggesting discrete anesthetic action sites. This method validates new ways to study volatile anesthetic-protein interactions.

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Area of Science:

  • Biochemistry
  • Pharmacology

Background:

  • Volatile anesthetics like halothane and isoflurane bind to serum albumin.
  • Previous methods (NMR, gas chromatography) showed saturable binding.
  • A novel photoaffinity labeling method was developed to study these interactions.

Purpose of the Study:

  • Validate a novel direct photoaffinity labeling method.
  • Determine halothane binding characteristics to serum albumin.
  • Extend the method to other soluble proteins to understand anesthetic-protein interactions.

Main Methods:

  • Used 14C-halothane and UV light for photoaffinity labeling of serum albumin, bacterial luciferase, poly-(L-lysine), and poly-(L-glutamate).
  • Quantified covalently bound label via scintillation counting after purification.

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  • Calculated binding parameters using nonlinear least-squares fitting.

    • Main Results:

    • Serum albumin exhibited saturable halothane binding (apparent KD 0.3–0.5 mM).
    • Other volatile anesthetics and ligands inhibited halothane binding to serum albumin.
    • Binding to bacterial luciferase and poly-(L-lysine) was largely nonsaturable at pH 7.
    • Increased alpha-helical conformation of poly-(L-lysine) led to saturable binding.

    Conclusions:

    • Photoaffinity labeling aligns with conventional methods for studying halothane binding.
    • This technique is valuable for investigating volatile anesthetic binding sites.
    • Halothane binding is protein- and conformation-dependent, with some interactions saturable at clinical concentrations.