Kehoe Joseph J, Brodkorb André, Mollé Daniel, Yokoyama Emilie, Famelart Marie-Héléne, Bouhallab Saíd, Morris Edwin R, Croguennec Thomas
Moorepark Food Research Centre, Teagasc, Fermoy, County Cork, Ireland.
J Agric Food Chem. 2007 Aug 22;55(17):7107-13. doi: 10.1021/jf070397r. 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.
本文采用一种新方法来确定在牛β-乳球蛋白A热变性过程中哪些巯基会暴露出来。加热后暴露的巯基用5-（（（（2-碘乙酰基）氨基）乙基）氨基）萘-1-磺酸（IAEDANS）进行封闭。结果表明，IAEDANS是一种合适的封闭剂，其在336nm处的吸光度能够通过凝胶渗透色谱法定量蛋白质混合物中暴露的巯基。结合在负离子模式下通过基质辅助激光解吸电离（MALDI）MS/MS对结合的IAEDANS进行特异性裂解，这有助于鉴定蛋白质酶解后含有被封闭半胱氨酸的肽段。在肽段的MALDI MS/MS分析中，在正离子模式下，IAEDANS分子仍与半胱氨酸结合，从而能够准确鉴定加热后哪些半胱氨酸被暴露。在β-乳球蛋白A中发现，无论加热时间长短，半胱氨酸66和半胱氨酸160主要都会暴露出来。
