Contributions of components in guanidine hydrochloride to hemoglobin unfolding investigated by protein film electrochemistry.

The contribution of the chloride anion (Cl(-)) and guanidinium cation (Gdn(+)) to the denaturing efficiency of guanidine hydrochloride (GdnCl) upon the unfolding of hemoglobin (Hb) was investigated electrochemically. Hb was entrapped in a didodecyldimethylammonium bromide (DDAB)-film-modified glassy carbon electrode and unfolded by the components of GdnCl. The changes of the direct electrochemical behaviors of Hb, including peak current (I(p)), formal potential (E(o)'), and peak-to-peak separation (DeltaE), were utilized to characterize different unfolded states of Hb. UV-vis, circular dichroism, and fluorescence spectroscopy were also used to verify the structural information of Hb during the unfolding process. The results indicated that the denaturing efficiency of GdnCl was contributed to by Gdn(+) and Cl(-) in synchronization, and the portions of such contributions were concentration-dependent. In addition, the presence of Gdn(+), Cl(-), or GdnCl can enhance the reversibility of the redox reaction of Hb to the same degree. The method provides not only an easy way to better understand the conformational changes of Hb but also a strategy to control its conformation.