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P. Di Simplicio, L. Lusini, F. Giannerini, D. Giustarini, A. Bellelli, G. Boumis, G. Amiconi and R. Rossi, “Nitric Oxide and the Cell,” In: S. Moncada, G. Nisticò, G. Bagetta and E. A. Higgs, Eds., Proliferation Differentiation and Death, Portland Press, Colchester, 1998, pp 47-59.

has been cited by the following article:

• TITLE: Quantification of Global Protein Disulfides and Thiol-Protein Mixed Disulfides to Study the Protein Dethiolation Mechanisms

  AUTHORS: Lucia Coppo, Raffaella Priora, Sonia Salzano, Pietro Ghezzi, Paolo Di Simplicio

  KEYWORDS: Protein Disulfides; S-Glutathionylation; Thiol pKa; Diamide; Oxidative Stress

  JOURNAL NAME: American Journal of Analytical Chemistry, Vol.4 No.10A, September 26, 2013

  ABSTRACT: The redox state of cellular thiols is widely studied because it was recently linked to many different diseases and pathologies. In this work we quantified the concentrations of protein disulfides (PSSP) and thiol-protein mixed disulfides (XSSP) in rat tissues (liver, kidney and heart) and cells (Raw 264.7) by an improved method of XSSP and PSSP determination after oxidative stress induced by diamide. Under native and denaturing conditions, a thiol block by N-ethymaleimide was introduced to avoid thiol exchange reaction activations by protein SH groups (PSH) (PSH + XSSP ←→ PSSP + XSH) and alterations of original XSSP/PSSP levels. Low molecular weight thiols (XSH) and PSH were respectively measured by HPLC on supernatants and on corresponding pellets by DTNB (Ellman’s reagent) after dithiothreitol reduction. PSSP concentrations of liver, heart and kidney were respectively 0.304, 0.605 and 0.785 μmoles/g and after diamide exposure they were significantly augmented of about 65%-70% in liver and heart, but not in the kidney. Normal XSSP, that were -20 times lower than normal PSSP were induced by diamide in liver and heart of about 40 times, but not in kidney. Thermodynamic criteria regarding the pKa values of thiols engaged as PSSP and GSSP were used to interpret dethiolation mechanisms via thiol exchange reactions.