Contrasting effects of pH on the modulation of the structural integrity of hemoglobin induced by sodium deoxycholate

Bile salt-mediated conformational modification of hemoglobin (Hb) was examined at three different pHs i.e., 3.2, 7.4 and 9.0. The added bile salt, sodium deoxycholate (NaDC), decreases the α-helicity in Hb (α-helix: 71.3% → 61.7% in the presence of 9.6 mM NaDC, and 83.2% → 66.2% in the presence of 14 mM NaDC, at pH 7.4 and 9.0, respectively), while a reverse pattern of modification in the Circular Dichroism (CD) spectra of Hb is found at pH 3.2. The acid-induced denatured Hb (pH 3.2) regains its structural integrity by changing conformation from a random coil to an α-helix rich secondary structure upon addition of NaDC (α-helix: 10.4% → 53.4%, β-sheet: 31.0% → 18.5% and random coil: 58.6% → 28.1%, in the presence of 0.65 mM NaDC). Also, a step-wise binding interaction pattern of Hb with NaDC was revealed at pH 7.4 and 9.0 upon variation of steady-state fluorescence intensity and average lifetime of Hb. From the fluorescence lifetime decay pattern, the decrement of energy transfer from Trp to a heme group was found upon the addition of NaDC at pH 7.4 and 9.0. However, at pH 3.2, the modification of the time-resolved fluorescence decay behavior of Hb within NaDC is typically reversed, where the energy transfer from Trp to heme is restored to some extent. Thermodynamic analysis suggests that the Hb-NaDC binding interaction is characterized by a dominant entropic contribution interpreted on the basis of release of ordered water molecules to the bulk aqueous phase.