title: Direct, simple derivatization of disulfide bonds in proteins with organic mercury in alkaline medium without any chemical pre-reducing agents.
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.
authors.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.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Campanella
authors.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.
authors.forename: Massimo
authors.initials: M
authors.lastname: Onor
authors.affiliation: 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.
authors.forename: Carlo
authors.initials: C
authors.lastname: Ferrari
authors.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.
authors.forename: Alessandro
authors.initials: A
authors.lastname: D'Ulivo
authors.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. Electronic address: bramanti@pi.iccom.cnr.it.
authors.forename: Emilia
authors.initials: E
authors.lastname: Bramanti
mesh_terms: D002850:Chromatography, Gel / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods; D004220:Disulfides / Q000032:analysis*; D006902:Hydroxymercuribenzoates / Q000737:chemistry*; D009113:Muramidase / Q000032:analysis*; D019163:Reducing Agents
chemical_list: D004220:Disulfides; D006902:Hydroxymercuribenzoates; D019163:Reducing Agents; C030601:4-hydroxymercuribenzoate; D009113:Muramidase
keywords: Alkaline medium; Atomic fluorescence spectrometry; Disulfide bond derivatization; Proteins; p-Hydroxymercuribenzoic acid