Characterization of the covalent binding of allyl isothiocyanate to β -lactoglobulin by fluorescence quenching, equilibrium measurement, and mass spectrometry

Julia Katharina Keppler*, Tomas Koudelka, Kalpana Palani, Mayra Christina Stuhldreier, Friedrich Temps, Andreas Tholey, Karin Schwarz

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

51 Citations (Scopus)

Abstract

Reversible binding of small compounds through hydrophobic interactions or hydrogen bonding to food proteins (e.g. milk proteins) is a thoroughly researched topic. In contrast, covalent interactions are not well characterized. Here, we report a rare form of positive-cooperativity-linear binding of allyl isothiocyanate with β-lactoglobulin, resulting in the cleavage of a disulfide bond of the protein. We compared three methods (i.e. fluorescence quenching, equilibrium dialysis, and headspace-water equilibrium) to characterize the binding kinetics and investigated the molecular binding by mass spectrometry. The methodologies used were found to be comparable and reproducible in the presence of high and low ligand concentrations for fluorescence quenching and equilibrium-based methods respectively.
Original languageEnglish
Pages (from-to)1103-1117
Number of pages15
JournalJournal of Biomolecular Structure and Dynamics
Volume32
Issue number7
DOIs
Publication statusPublished - 3 Jul 2014
Externally publishedYes

Keywords

  • allyl isothiocyanate
  • covalent ligand binding
  • fluorescence quenching
  • tryptic digestion
  • β-lactoglobulin

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