Concentration of Denaturant at Midpoint of Unfolding Transition (Cm)
Fluorescence Intensity , Urea Denaturation
Denaturation measurements were performed by mixing appropriate proportions of stock denaturant solution (e.g., 7.5 M GdnHCl, 0.2 M NaCl, 20 mM sodium acetate, pH 5.0), buffer solution (e.g., 0.2 M NaCl, 20 mM sodium
acetate, pH 5.0), and protein solution (e.g., 1 mg/mL ?1, dialyzed exhaustively against 0.2 M NaCl, 20 mM sodium acetate, pH 5.0), followed by incubation at 25 °C for 4-12 h and spectral determination. Unfolding measurements involving the use of urea were completed within 24 h after preparation of the urea solutions.
Fluorescence measurements were conducted using a SPEX FluoroMax spectrofluorometer. Fluorescence intensity (normalized to the lamp intensity) was monitored at excitation wavelengths of 275 and 295 nm, and emission at 340 nm, with excitation and emission bandwidths of 3-5 nm, depending on the protein concentration (about 5-10 µg/mL). Fluorescence recordings were made usinga1cm path length cuvette, maintaining temperature at 25.0 ( 0.1 °C. Corrections were made for buffer and denaturant contributions to the fluorescence intensity