An electrochemical approach based on protein controllable unfolding was developed and applied in combination with in-situ reaction in order to investigate the electrocatalytic pathway for hemoglobin (Hb) toward nitric oxide (NO). Hb was entrapped in a dimethyldidodecylammonium bromide (DDAB) film modified glassy carbon electrode (DDAB/Hb/GCE). Two typical denaturants of acid and urea were synergistically utilized to control the incorporated Hb to a most unfolded state without losing heme groups. Under optimal conditions, the unfolded DDAB/Hb/GCE exhibited accelerated direct electron transfer. The sensitivities for the detection of ascorbic acid (AA), NaNO(2) and NO were improved as 3, 10 and 12 times higher than those on the native DDAB/Hb/GCE, and the limits of detection (LODs) for AA, NaNO(2) and NO were down to 0.33, 0.83 and 0.063 μM, respectively. The unfolded DDAB/Hb/GCE was further applied for the investigation of Hb-NO interaction in NaNO(2) solution. With successive additions of AA, NO was generated in situ on DDAB/Hb/GCE. A new reduction peak of the intermediate HbFe(II)-HN(2)O(2) was successfully revealed near -0.65 V. The whole electrocatalytic mechanism was proposed and verified by density functional theory. The method can be a promising platform for facile study of the interaction between NO and heme proteins.
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