Fig 3.

High pH denaturation of Cyt c (no urea). (A) Above pH 12, denaturation occurs in two steps with a populated intermediate. A partial unfolding removes 45% of the CD₂₂₂ of the native protein and recovers all of the fluorescence of the U state (corrected for Trp pH titration and quenching). Both probes show the same midpoint (pK 12.8) and slope (1.5). Equilibrium unfolding to U occurs above pH 13. (B) The kinetics of intermediate formation. The black curve in Fig. 5A is constructed from Fl data in 1.3 M urea. The zero urea data are shown here because CD₂₂₂ is obscured in high pH urea.