Thermodynamic characterization of cytochrome c at low pH. Observation of the molten globule state and of the cold denaturation process.

作者: Yutaka Kuroda , Shun-ichi Kidokoro , Akiyoshi Wada

DOI: 10.1016/0022-2836(92)90265-L

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摘要: Abstract Several reports have pointed out the existence of intermediate states (both kinetic and equilibrium intermediate) between native denatured states. The molten globule state, a compact state in which secondary structure is formed but tertiary fluctuates considerably, currently being studied intensively because its possible implication folding process several proteins. We examined thermal stability horse cytschrome c at low pH 2.0 3.2 different potassium chloride concentrations by absorbance Soret band, far near-ultraviolet circular dichroism (u.V. c.d.) tryptophan fluorescence using multidimensional spectrophotometer. concentration ranged from 0 m to 0.5 . experimental denaturation curves show that: (1) helical content cytochrome remains stable higher temperature when salt increased; whereas (2) extent ordering weakly dependent on concentration; (3) for , stabilization induced binding anions. Other salts such as NaCl, LiCl, ferricyanide (K 3 Fe(CN) 6 ) Na 2 SO 4 may also be used stabilize state. thermodynamic analysis c.d. 222 nm 282 shows that, two-state (native denatured) transition observed low-salt concentration, near-u.v. melting do not coincide with each other high-salt minimum three (IIb, or IIc, necessary achieve sufficient analysis. (called IIc) attributed high absence structure. Therefore, pH, present least four (native, IIb, IIc depending temperature. parameters, i.e. Gibbs free energy differences ( ΔG ), enthalpy (Δ H midpoint temperatures T (IIb → (IIc) are determined. give estimates heat capacity C p dependence differences. change difference non-zero. number charges (protons anions) released upon transitions determined analysing anion energy. At these values good agreement microcalorimetric study can explain cold sub-zero temperature, was first study. Finally, phase diagram proposed previously Ohgushi Wada re-examined view data.

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