In this work we have studied the derivatization of protein disulfide bonds with p-Hydroxymercurybenzoate (pHMB) in strong alkaline medium without any preliminary reduction. The reaction has been followed by the determination of the protein-pHMB complex using size exclusion chromatography coupled to a microwave/UV mercury oxidation system for the on-line oxidation of free and protein-complexed pHMB and atomic fluorescence spectrometry (SEC-CVG-AFS) detection. The reaction has been optimized by an experimental design using lysozyme as a model protein and applied to several thiolic proteins. The proposed method reports, for the first time, that it is possible to label 75-100% cysteines of proteins and, thus, to determine thiolic proteins without the need of any reducing step to obtain reduced SH groups before mercury labelling. We obtained a detection limit of 100 nmol L(-1) based on a signal-to-noise ratio of 3 for unbound and complexed pHMB, corresponding to a detection limit of proteins ranged between 3 and 360 nmol L(-1), depending on the number of cysteines in the protein sequence.
Keywords: Alkaline medium; Atomic fluorescence spectrometry; Disulfide bond derivatization; Proteins; p-Hydroxymercuribenzoic acid.
Copyright © 2014 Elsevier B.V. All rights reserved.