Figure 3

Reaction of rLF toward GSSG and natural disulfides. (A) Disappearance of rLF cysteines (1 µM, 32 µM protein –SH) during the reaction with 1 mM GSSG in 0.2 M urea, 10 mM sodium acetate buffer pH 5.0, at 25 °C. The 32 –SH/mole of rLF were determined with DTNP. The error bars represent the SD from three independent experiments. The first phase of reaction (up to 20 min) when only a few cysteines reacted with GSSG is reported in the Inset. (B) Second order kinetic constants of cysteines (k_{rLF SH}) normalized to the rate constants for the free GSH or, only for GSSG, normalized to the constant calculated for an unperturbed protein cysteine (k_{free thiol}). The number of protein cysteine per mole with a given reactivity is reported on the top of each column. Error bars represent the propagation of uncertainties for the quotients²¹. (C) 3D structure of native rLF (PDB id: 1LFG)²⁵ is depicted in pink ribbons with cysteines in yellow sticks. The four most reactive cysteines toward GSSG identified by MS analysis are displayed in four different orange palettes (spheres). The amino acid number in black derived from X-ray structure in PDB database. The 3D representation is obtained by UCSF chimera software²⁶. (D) Peak area values of the four glutathionylated tryptic peptide fragments after reaction with GSSG (see Materials and methods for details) as revealed by the eXtracted Ion Current procedure. No other modified fragments were detected. Error bars represent the SD from three analytical replicate LC–MS analyses. (E) Quenching of the intrinsic fluorescence of rLF (1.1 µM) after addition of variable GSSG concentrations (from 0.2 to 4 mM) in 0.2 M urea, 10 mM sodium acetate buffer pH 5.0 at 25 °C. Each point was subtracted by the fluorescence of NATA with GSSG under the same conditions. Measurements were made immediately after mixing to avoid any glutathionylation interference. The error bars represent the SD from three independent experiments. (F) Schematic representation of the reaction of rLF with GSSG.