Identification of the binding of β-lactoglobulin (β-Lg) with sulfhydryl (–SH) blocking reagents by polyacrylamide gel electrophoresis (PAGE) and electrospray ionisation/time of flight-mass spectrometry (ESI/TOF-MS)

Abstract

β-Lactoglobulin (β-Lg) is relatively unstable during thermal processes used in the dairy industry. One of many ways to improve the heat stability of β-Lg is by chemical modification by protein–reagent binding. Potential reagents, such as 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and N-ethylmaleimide (NEM), bind to the exposed free sulfhydryl (–SH) group of denatured β-Lg and prevent it from being involved in protein aggregation reactions. However, no study has shown the specific binding of these reagents to β-Lg. Furthermore, the question of how many molecules of these reagents bind to β-Lg remains unanswered. In this research, the reactions of DTNB or NEM with the reactive monomers of β-Lg were identified by the presence of bands for 5-thio-2-nitrobenzoyl (TNB)- or NEM-modified monomers on Native-PAGE and Non-Reducing (NR) sodium deodecyl sulphate (SDS)-PAGE gels, and by an additional mass of β-Lg monomers corresponding to a molecule of TNB or NEM in mass spectra.