TY - JOUR
T1 - Characterization of the covalent binding of allyl isothiocyanate to β -lactoglobulin by fluorescence quenching, equilibrium measurement, and mass spectrometry
AU - Keppler, Julia Katharina
AU - Koudelka, Tomas
AU - Palani, Kalpana
AU - Stuhldreier, Mayra Christina
AU - Temps, Friedrich
AU - Tholey, Andreas
AU - Schwarz, Karin
PY - 2014/7/3
Y1 - 2014/7/3
N2 - 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.
AB - 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.
KW - allyl isothiocyanate
KW - covalent ligand binding
KW - fluorescence quenching
KW - tryptic digestion
KW - β-lactoglobulin
U2 - 10.1080/07391102.2013.809605
DO - 10.1080/07391102.2013.809605
M3 - Article
C2 - 23808652
AN - SCOPUS:84899946156
SN - 0739-1102
VL - 32
SP - 1103
EP - 1117
JO - Journal of Biomolecular Structure and Dynamics
JF - Journal of Biomolecular Structure and Dynamics
IS - 7
ER -