Interaction of phenazinium-based photosensitizers with the 'N' and 'B' isoforms of human serum albumin: Effect of methyl substitution.

The present work is focused on exploring the interaction of two phenazinium-based biological photosensitizers, phenosafranin (PSF) and safranin-O (SO), with human serum albumin (HSA), with particular emphasis on the physiologically significant NB conformational transition of the protein on the dye:HSA interaction. In addition, the presence of methyl substitution on the planar phenazinium ring in SO paves way for looking into the effect of simple chemical manipulation (that is, methyl substitution on the dye nucleus) on the dye:protein interaction behavior as a function of various (pH-induced) isoforms of HSA. Our results reveal a significantly stronger binding interaction of SO with the B isoform of HSA (at pH9.0) compared to that with the N isoform (at pH7.4). On the contrary, the PSF:HSA interaction is found to be reasonably insensitive to the aforesaid conformational transition of HSA. However, the probable binding location of both the dye molecules (PSF and SO) is found to be within the protein scaffolds (domain IB). This is further quantified from the modulation of fluorescence decay behavior of the dyes within the protein scaffolds. It is important to note that the rotational relaxation behavior of the protein-bound dyes reveals an unusual 'dip-rise-dip', an observation not reported earlier. Such unusual anisotropy decay is meticulously analyzed by an associated (or multicomponent) exponential decay model which emphasizes on the fractional contributions from differential classes of fluorophore populations characterized by the fast (due to unbound or solvent exposed part of the fluorophore) and slow (due to embedded or bound part) motions, in combination with their different local mobilities. Furthermore, the translational diffusion of the dye molecules in the presence of the protein in different isoforms (N-form or B-form) at a single molecule level is also measured by Fluorescence Correlation Spectroscopy (FCS).