Binding interaction of phenazinium-based cationic photosensitizers with human hemoglobin: Exploring the effects of pH and chemical structure

The present study demonstrates a spectroscopic study on the interaction of two phenazinium-based cationic photosensitizers, namely, phenosafranin (PSF) and safranin-O (SO) with human hemoglobin (Hb) with particular emphasis on exploring the effects of pH and chemical structures of the dye molecules on the binding phenomenon. The protein (Hb) undergoes complex conformational transitions depending on the medium pH. The dye molecules exhibit a prominent fluorescence quenching following interaction with Hb under various experimental conditions (pH 3.5, 7.4, and 9.0). Our combined steady-state and time-resolved spectroscopic results provide persuasive evidence for static quenching mechanism showing that the dye:Hb interaction proceeds through ground-state complex formation. The meticulous investigations on the pH-dependence of the interaction of the dye molecules with the protein reveal a relatively strong binding of PSF as well as SO with Hb at physiological pH and alkaline pH, while the binding is weaker at acidic pH at which Hb predominantly exists as monomeric units. The binding constant for PSF:Hb interaction is K(PSF:Hb) = (1.09 ± 0.06) × 10⁶ M⁻¹ and that of SO:Hb interaction is K(SO:Hb) = (1.34 ± 0.07) × 10⁵ M⁻¹ at pH 7.4. However, at pH 3.5, the binding constant values are K(PSF:Hb) = (3.58 ± 0.18) × 10⁴ M⁻¹ and K(SO:Hb) = (4.29 ± 0.22) × 10⁴ M⁻¹ and at pH 9.0, the values are K(PSF:Hb) = (8.08 ± 0.40) × 10⁴ M⁻¹ and K(SO:Hb) = (5.07 ± 0.25) × 10⁴ M⁻¹. This depicts a much stronger binding interaction of the dyes with the native Hb at pH 7.4 compared to those at pH 3.5 and 9.0. Our results also unveil the effect of chemical structures of the dyes on the interaction phenomenon in the sense that the binding constant of PSF with Hb is found to be higher than that of SO at pH 7.4 and pH 9.0. The present study also focuses on exploring such important aspects of the interaction phenomena as the effect of binding of the dyes on the protein conformation by circular dichroism spectroscopy and probable binding location of the dyes within the protein scaffolds via micropolarity measurements and molecular docking simulation.