Fig. 1.

(A) Effect of 1 nM and 100 μM cyanide (CN) on the respiration of rotenone- and antimycin-treated mouse liver mitochondria by using the TMPD–ascorbate system. Briefly, 20 mg of purified mitochondria were incubated with 1 nM or 100 μM KCN for 15 min in resuspension buffer (75 mM sucrose, Hepes 20 mM, 50 mM KCl, 1 mM NaH₂PO₄, 1 mM MgCl₂, and 1 mM EGTA, pH 7.4). The respiratory activity of mouse liver mitochondria was measured with a Clark-type electrode, in an all-glass reaction chamber magnetically stirred, at 25 °C. Where indicated, 5 μM rotenone/1 μM antimycin A and 0.4 mM TMPD/5 mM ascorbate were added. The rate of O₂ consumption (in units of nanomoles of O₂ per minute per mg of protein) was calculated 10 to 14 s after the TMPD/ascorbate injection. Values are mean ± SEM; statistics of control vs. 1 nM is are nonsignificant while control vs. 100 μM KCN is P ≤ 0.01, one-way ANOVA. (B) Time course of scission of the SS bond in 20 μM DTNB (Ellman’s reagent) by 2 mM total cyanide at 25 °C. The buffer used was 0.1 M Na-phosphate, pH 7. The data are represented as difference spectra relative to DTNB alone. The data were filtered by singular value decomposition (6) and reconstructed with 2 s values (s₁ = 4.3483, s₂ = 0.4910, s₂ = 0.0438). Analogous time courses were determined at 5, 8, and 11 mM total HCN. Time courses at 412 nm (slice through difference spectra) were fitted to an exponential process [$y=\alpha \left(1-e^{-k_{obs}t}\right)$]. A plot of k_obs, measured at different cyanide concentrations, was linear, with a slope equal to the second-order rate constant reported herein. All calculations were carried with MathWorks MATLAB.