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Study of the disulfide reduction of denatured proteins by liquid chromatography coupled with on-line cold-vapor-generation atomic-fluorescence spectrometry (LC–CVGAFS)

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Abstract

Hydrophobic-interaction chromatography coupled on-line with chemical-vapor-generation atomic-fluorescence spectrometry (HIC–CVGAFS), optimized recently for the analysis of thiol-containing proteins under denaturing conditions, has been used to study the chemical reduction of denatured proteins. Four proteins chosen as models (human serum albumin (HSA), bovine serum albumin (BSA), α-lactalbumin (α-Lac) from bovine milk, and lysozyme from chicken egg (Lys)) were denatured with urea and reduced with dithiothreitol (DTT), with selenol as catalyst. The method is based on derivatization of the –SH groups of proteins with p-hydroxymercurybenzoate (PHMB), followed by HIC separation and post-column on-line reaction of the derivatized reduced, denatured proteins with bromine generated in situ. HgII, derived from rapid conversion of uncomplexed and protein-complexed PHMB, is selectively detected by AFS in an Ar/H2 miniaturized flame after sodium borohydride (NaBH4) reduction to Hg°. The yield of the reduction was studied as a function of reductant concentration, reduction time (tred), and urea concentration. Results showed that the optimum values for DTT and selenol concentrations and for tred were between 1 and 100 mmol L−1 and between 1 and 20 min, respectively, depending on the protein studied. The percentage disulfide bond reduction increases as the urea concentration used for protein denaturation increases, giving a single-step sigmoid increment for single-domain, low-MW proteins (α-Lac and Lys), and a two-step sigmoid increment for multi-domain, high MW proteins (HSA and BSA). The shapes of plots of percentage reduced disulfide against urea concentration are characteristic of each protein and are correlated with the location of S–S in the protein. Under the adopted conditions complete protein denaturation is the conditio sine qua non for obtaining 100% S–S reduction. The detection limit for denatured, reduced proteins examined under the optimized conditions was found to be in the range 1–5×10−12 mol L−1 (10–30 pg), depending on the protein considered.

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Acknowledgments

This work was financially supported by CNR and Ambiente s.c.r.L. (Carrara, MS, Italy; DOCUP 2000–2006, Regione Toscana). The authors would like to thank ThermoQuest, for providing part of the instrumentation, and Mrs M. Cempini, C. Lanza and M.C.M. Mascherpa for their technical support.

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Authors and Affiliations

  1. Laboratory of Instrumental Analytical Chemistry, Italian National Research Council-Istituto per I Processi Chimico-Fisici, Via G. Moruzzi 1, 56124, Pisa, Italy

    Emilia Bramanti, Cristina Lomonte, Massimo Onor, Giorgio Raspi & Alessandro D’Ulivo

  2. Department of Chemistry and Industrial Chemistry, University of Pisa, Via Risorgimento 35, 56126, Pisa, Italy

    Roberto Zamboni

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  1. Emilia Bramanti
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  2. Cristina Lomonte
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  3. Massimo Onor
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  4. Roberto Zamboni
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  5. Giorgio Raspi
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  6. Alessandro D’Ulivo
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Correspondence to Emilia Bramanti.

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Bramanti, E., Lomonte, C., Onor, M. et al. Study of the disulfide reduction of denatured proteins by liquid chromatography coupled with on-line cold-vapor-generation atomic-fluorescence spectrometry (LC–CVGAFS). Anal Bioanal Chem 380, 310–318 (2004). https://doi.org/10.1007/s00216-004-2746-3

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  • DOI: https://doi.org/10.1007/s00216-004-2746-3

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