J 815 cd

Samples were prepared using a Yfh1 concentration of 10 μM in a 10 mM HEPES buffer at pH 7.0 and with varying concentrations of PEG 20, dextran 40, Ficoll 70 or Ficoll 400. Baseline correction was obtained by subtraction of the appropriate buffer spectrum. Thermal unfolding curves were obtained by monitoring the ellipticity at 220 nm using a Jasco J-815 CD spectropolarimeter equipped with a Jasco CDF-4265/15 Peltier unit. The 1 mm path length cells (Hellma) were used and a heating rate of 2 °C min⁻¹ in the temperature range 2–70 °C.

UV spectra were recorded using a JASCO J-815 CD spectropolarimeter (Jasco Inc., Easton, MD, USA). For this study, solutions of compound 5 were prepared in 25 mM sodium phosphate buffer (pH 7.4) across a concentration range of 0.016–0.5 mM. Background correction was applied to all spectra by subtracting the appropriate blank. UV experiments were conducted in duplicate.

Far-UV CD spectra were recorded on a Jasco J-710 spectropolarimeter. Samples were prepared using a Yfh1 concentration of 10 µM in 10 mM HEPES buffer at pH 7.5 and with a variety of salts: NaCl (Sigma-Adrich), KCl (Aldrich), MgCl₂ (Aldrich), CaCl₂ (J.T.Baker), NaF (May&Baker), NaH₂PO₄ (Carlo Erba), Na₂SO₄ (Sigma-Aldrich) and NaI (Carlo Erba). Baseline correction was performed by subtraction of the appropriate buffer spectrum. Thermal unfolding curves were obtained by monitoring the ellipticity at 220 nm using a Jasco J-815 CD spectropolarimeter equipped with a Jasco CDF-4265/15 Peltier unit. Measurements were repeated at least twice on independent protein preparations to ensure reproducibility of the results. All samples used Yfh1 at 10 µM in a 10 mM HEPES buffer at pH 7.5 and varying concentrations of NaCl, KCl (Fisher Scientific), MgCl₂, CaCl₂, FeSO₄, NaF (Sigma-Aldrich), NaH₂PO₄ (Acros Organics) and Na₂SO₄ (Merck). 2 mm path length cuvettes (Hellma) were used with a heating rate of 2°C/min in the temperature range 0–80°C. In the case of the ferrous salt rigorous anaerobic conditions were achieved by preparing the samples in a glove box, sealing the cuvette and performing the measurements under N₂ (g) flow.