Direct, simple derivatization of disulfide bonds in proteins with organic mercury in alkaline medium without any chemical pre-reducing agents

• A simple procedure for the derivatization of proteins disulfide bonds.
• Cysteine groups in several proteins derivatised with pHMB in alkaline media.
• 75–100% labelling of cysteines in proteins with pHMB.

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.

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