| 17650000 |
Kehoe JJ, Brodkorb A, Molle D, Yokoyama E, Famelart MH, Bouhallab S, Morris ER, Croguennec T: Determination of exposed sulfhydryl groups in heated beta-lactoglobulin A using IAEDANS and mass spectrometry. J Agric Food Chem. 2007 Aug 22;55(17):7107-13. Epub 2007 Jul 25.
This paper takes a new approach to determining which sulfhydryl groups are exposed during the heat denaturation of bovine beta-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 beta-lactoglobulin A it was found that cysteine 66 and cysteine 160 were predominantly exposed regardless of the length of exposure to heat. |
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