Skip to Content
MilliporeSigma
  • Hydration-state change of horse heart cytochrome c corresponding to trifluoroacetic-acid-induced unfolding.

Hydration-state change of horse heart cytochrome c corresponding to trifluoroacetic-acid-induced unfolding.

Biophysical journal (2013-01-22)
Yusuke Miyashita, Tetsuichi Wazawa, George Mogami, Satoshi Takahashi, Yoshihiro Sambongi, Makoto Suzuki
ABSTRACT

We investigate the hydration state of horse-heart cytochrome c (hh cyt c) in the unfolding process induced by trifluoroacetic acid (TFA). The conformation of hh cyt c changes from the native (N) state (2.9 < pH < 6.0) to the acid-unfolded (U(A)) state (1.7 < pH < 2.0) to the acid-induced molten globule (A) state (pH ∼1.2). Hydration properties of hh cyt c during this process are measured at 20°C by high-resolution dielectric relaxation (DR) spectroscopy, UV-vis absorbance, and circular dichroism spectroscopy. Constrained water of hh cyt c is observed at every pH as an ∼5-GHz Debye component (DC) (DR time, τ(D) ∼30 ps) and its DR amplitude (DRA) is increased by 77% upon N-to-U(A) transition, when pH changes from 6.0 to 2.0. Even in the N state, the DRA of the constrained-water component is found to be increased by 22% with decreasing pH from 6.0 to 2.9, suggesting an increase in the accessible surface area of native hh cyt c. Moreover, hypermobile water around native hh cyt c is detected at pH 6.0 as a 19-GHz DC (τ(D) ∼ 8.4 ps <τ(DW) = 9.4 ps), but is not found at other pH values. The DRA signal of constrained water is found to return to the pH 2.9 (N-state) level upon U(A)-to-A transition. Fast-response water (slightly slower than bulk) around A-state hh cyt c is detected at pH 1.2, and this suggests some accumulation of TFA(-) ions around the peptide chain. Thus, this high-resolution DR spectroscopy study reveals that hh cyt c exhibits significant hydration-state change in the TFA-unfolding process.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Trifluoroacetic acid, SAJ special grade, ≥99.0%
Supelco
Trifluoroacetic acid, analytical standard
Sigma-Aldrich
Trifluoroacetic acid, suitable for HPLC, ≥99.0%
Sigma-Aldrich
Trifluoroacetic acid, ReagentPlus®, 99%
Sigma-Aldrich
Trifluoroacetic acid, puriss. p.a., suitable for HPLC, ≥99.0% (GC)
Sigma-Aldrich
Trifluoroacetic acid, ≥99%, for protein sequencing