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

Year: 2014

Authors: Campanella B., Onor M., Ferrari C., D’Ulivo A., Bramanti E.

Autors Affiliation: National Research Council of Italy, C.N.R., Istituto di Chimica dei Composti Organo Metallici-ICCOM- UOS Pisa, Area di Ricerca, Via G. Moruzzi 1, 56124 Pisa, Italy; National Research Council of Italy, C.N.R., Istituto Nazionale di Ottica, INO-UOS Pisa, Area di Ricerca, Via G. Moruzzi 1, 56124 Pisa, Italy

Abstract: 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.

Journal/Review: ANALYTICA CHIMICA ACTA

Volume: 843      Pages from: 1  to: 6

KeyWords: Alkaline medium; Atomic fluorescence spectrometry; Disulfide bond derivatization; P-Hydroxymercuribenzoic acid; Proteins
DOI: 10.1016/j.aca.2014.07.003

Citations: 7
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