Direct electron transfer for hemoglobin in biomembrane-like dimyristoyl phosphatidylcholine films on pyrolytic graphite electrodes

Stable thin films made from dimyristoyl phosphatidylcholine (DMPC) with incorporated hemoglobin (Hb) on pyrolytic graphite (PG) electrodes were characterized by electrochemical and other techniques. Cyclic voltammetry (CV) of Hb-DMPC films showed a pair of well-defined and nearly reversible peaks at about −0.27 V vs. saturated calomel electrode (SCE) at pH 5.5, characteristic of Hb heme Fe(III)/Fe(II) redox couple. The electron transfer between Hb and PG electrodes was greatly facilitated in DMPC films. Apparent heterogeneous rate constants (k_s) were estimated by fitting square wave voltammograms of Hb-DMPC films to a model featuring thin layer behavior and dispersion of formal potentials for redox center. The formal potential of Hb heme Fe(III)/Fe(II) couple in DMPC films shifted linearly between pH 4.5 to 11 with a slope of −48 mV pH⁻¹, suggesting that one proton is coupled to each electron transfer in the electrochemical reaction. Soret absorption band positions suggest that Hb retains a near native conformation in DMPC films at medium pH. Differential scanning calorimetry (DSC) showed the phase transition for DMPC and Hb-DMPC films, suggesting DMPC has an ordered multibilayer structure. Trichloroacetic acid (TCA) was catalytically reduced by Hb-DMPC films with significant decreases in the electrode potential required.