Structural and Thermodynamic Studies on the Interaction of Iminium and Alkanolamine Forms of Sanguinarine with Hemoglobin

Binding of the iminium and alkanolamine forms of the benzophenanthridine anticancer alkaloid sanguinarine to hemoglobin (Hb) was investigated by absorbance, fluorescence, and circular dichroism spectral techniques, and by calorimetry. The binding affinity of the charged iminium was found to be of the order of 10(6) M-1, higher by one order than that of the neutral alkanolamine, from the analysis of the absorbance data. The fluorescence spectral data revealed that the quenching of Hb fluorescence by both forms of sanguinarine is due to the formation of a complex in the ground state and is of an unusual, static nature. Thermodynamic data revealed that the binding of the iminium form was exothermic in nature while that of the alkanolamine was endothermic; the former case predominantly involved electrostatic and hydrogen bonding interactions but the latter was dominated by mostly hydrophobic interactions. Calculation of the molecular distances (r) between the donor (beta-Trp37) and acceptor (iminium and alkanolamine) according to Forster's theory suggests both forms of the alkaloid to be bound close to beta-Trp37 at the alpha 1 beta 2 interface of the protein. The iminium form induced greater secondary structural changes in Hb than the alkanolamine as revealed by synchronous fluorescence, circular dichroism and three-dimensional fluorescence spectroscopic studies. These results are consistent with a stronger binding of the iminium over the alkanolamine form. Nevertheless, the hydrophobic probe ANS was displaced from hemoglobin more easily by the alkanolamine form than by the iminium. The study showed that Hb binds more strongly to the biologically active iminium form than the alkanolamine, in contrast to the stronger binding of the latter to plasma protein serum albumin. Overall, this study presents insights on the interaction dynamics and energetics of the binding of the two forms of sanguinarine to hemoglobin.