FIG. 4.

The oxidative properties of NaHS and Na₂S solutions are due to polysulfide formation. (A) UV/Vis spectra of NaHS (from Sigma or Cayman) and Na₂S (from Alfa Aesar) dissolved freshly to 150 mM in 200 mM Tris-HCl, pH 8.0, were recorded and compared to that of 0.83 mg/ml K₂S_x dissolved in 200 mM Tris-HCl, pH 9.2. Absorbance peaks at 300 and 372 nm (indicated by arrows) are characteristic of polysulfides. (B) The stock solution of K₂S_x used in (A) was subjected to cyanolysis as described in Materials and Methods to determine sulfane sulfur (S⁰) levels. Data are given for 13.2 mg/ml K₂S_x. Preincubation of K₂S_x with 200 mM DTT for 10 min abolished the signal due to polysulfide degradation. Bars denote mean±range of duplicate wells. (B′) Activity of PTEN-WT (380 nM) was recorded upon injection of dilutions of the same K₂S_x stock solution. S⁰ concentrations given in parentheses are calculated from the cyanolysis data in (B). Curves represent means of triplicate wells. (C) Sulfane sulfur (S⁰) levels of the same NaHS and Na₂S solutions as in (A) were determined by cyanolysis. Data are given for 120 mM stock solutions. Bars denote mean±range of duplicate wells. (C′) PTEN activity was measured upon addition of buffer (untreated) or 10 μM of these NaHS and Na₂S solutions (injection indicated by arrow). Curves represent means of triplicate samples. Quantification of the effect of these agents on PTEN activity (expressed as PTEN inhibition) was done as for Figure 2B and is shown in (C′′). Bars denote means±SD of triplicate wells. The experiment shown in (A–C′′) was repeated twice. (D) 5 mM of NaHS (Cayman) or Na₂S, or 0.55 mg/ml of K₂S_x were pretreated with (D′) or without (D) 50 mM cyanide (KCN) for 1 h before they were injected into a running PTEN activity assay to give a final concentration of 50 μM NaHS/Na₂S±500 μM KCN or 5.5 ng/ml K₂S_x±500 μM KCN. Curves represent means of triplicate samples. The same protective effect by KCN was observed for roGFP2 (not shown). K₂S_x, potassium (poly)sulfide; Na₂S, sodium sulfide. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars