Second harmonic generation study of myoglobin and hemoglobin and their protoporphyrin IX chromophore at the water/1,2-dichloroethane interface

Second harmonic generation (SHG) from the proteins myoglobin (Mb) and hemoglobin (Hb) adsorbed at the interface between 1,2-dichloroethane (DCE) and a pH 7 aqueous buffer solution is reported and compared to the response from the Mb and Hb chromophore Fe(III) protoporphyrin IX (PpIX) freely adsorbed at the interface. Resonant SHG experiments were performed probing the PpIX π–π* transition and large SHG signals were obtained for bulk aqueous concentrations of proteins as low as 2 μM for Mb and 350 nM for Hb. Both protein adsorption isotherms demonstrated interactions between the proteins at the interface. A Gibbs free energy of adsorption of −82 ± 8 kJ mol−1 was determined for Mb and of −121 ± 12 kJ mol−1 for Hb at high surface coverage. From a light polarization analysis of the SHG signal, the angle of orientation of the free PpIX chromophore at the water/DCE interface was found to be 43 ± 2° with respect to the interface normal direction, assuming a Dirac delta function for the angle distribution, and 55 ± 2° for Mb, independently of the protein surface coverage. The bulk aqueous buffer solution pH was varied and a clear modification in the Hb monolayer was observed at a pH of 4, possibly the protonation of the PpIX carboxylic groups.