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.