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Determination of exposed sulphydryl groups in heated β-lactoglobulin A using IAEDANS and mass spectrometry
Kehoe, Joseph James Brodkorb, Andre Molle, Daniel Yokoyama, Emilie Famelart, Marie-Helene Bouhallab, Said Morris, Edwin R Croguennec, Thomas
Kehoe, Joseph James
Brodkorb, Andre
Molle, Daniel
Yokoyama, Emilie
Famelart, Marie-Helene
Bouhallab, Said
Morris, Edwin R
Croguennec, Thomas
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25/07/2007
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Joseph J. Kehoe, André Brodkorb, Daniel Mollé, Emilie Yokoyama, Marie-Héléne Famelart, Saíd Bouhallab, Edwin R. Morris, and Thomas Croguennec. Determination of Exposed Sulfhydryl Groups in Heated β-Lactoglobulin A Using IAEDANS and Mass Spectrometry. J. Agric. Food Chem., 2007, 55 (17), 7107–7113. DOI: 10.1021/jf070397r
Abstract
This paper takes a new approach to determining which sulfhydryl groups are exposed during the heat denaturation of bovine β-lactoglobulin A. The sulfhydryl groups exposed after heating were blocked with 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS). The results show that IAEDANS is a suitable blocking agent, and its absorbance at 336 nm enabled the quantification of exposed sulfhydryl groups in a mixture of protein species by gel permeation chromatography. Combined with the specific fragmentation of bound IAEDANS by matrix-assisted laser desorption ionization (MALDI) MS/MS in negative ionization mode, this facilitated the identification of peptides that contained blocked cysteines after enzymatic digestion of the protein. During MALDI MS/MS of the peptides, in positive ionization mode, the IAEDANS molecule remained bound to the cysteines, making it possible to identify exactly which cysteine had been exposed after heating. In β-lactoglobulin A it was found that cysteine 66 and cysteine 160 were predominantly exposed regardless of the length of exposure to heat.