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Related Experiment Videos

An active-site titration method for lipases.

D Rotticci1, T Norin, K Hult

  • 1Department of Chemistry, Organic Chemistry, Royal Institute of Technology, SE-100 44, Stockholm, Sweden.

Biochimica Et Biophysica Acta
|December 22, 1999
PubMed
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A new method accurately measures active lipase sites using irreversible inhibition. This technique works for soluble and immobilized lipases in various conditions, including organic solvents.

Area of Science:

  • Biochemistry
  • Enzymology

Background:

  • Lipases are crucial enzymes with diverse applications.
  • Accurate determination of active enzyme sites is essential for understanding enzyme kinetics and function.
  • Interfacial activation significantly impacts lipase activity and requires specific assay conditions.

Purpose of the Study:

  • To develop and validate a reliable method for active-site titration of lipases.
  • To assess the applicability of the method across different lipase types and states (soluble vs. immobilized).
  • To investigate the method's performance in various media, including aqueous and organic solvents.

Main Methods:

  • Active-site titration using irreversible inhibition with methyl p-nitrophenyl n-hexylphosphonate.
  • Application to five lipases with varying degrees of interfacial activation.

Related Experiment Videos

  • Titration in aqueous media with and without sodium dodecyl sulfate.
  • Adaptation for immobilized lipases in apolar organic solvents, including extraction of reaction byproducts.

    • Main Results:

    • The developed method successfully determined active sites for both soluble and immobilized lipases.
    • Lipases exhibiting pronounced interfacial activation required low concentrations of sodium dodecyl sulfate for accurate titration.
    • Adsorption of p-nitrophenolate by carriers of immobilized enzymes was overcome by extraction.
    • The method was successfully extended to immobilized lipases in apolar organic solvents.

    Conclusions:

    • The irreversible inhibition method provides a robust approach for active-site titration of lipases.
    • The method is versatile, applicable to diverse lipase preparations and reaction environments.
    • Optimization of assay conditions, such as detergent use and byproduct extraction, ensures reliable results.