Interaction of heme proteins with anionic polyfluorene: insights into physiological effects, folding events, and...

Because of the toxicity caused by the heme redox-active iron proteins, their elevated levels, localization, and accumulation in the brain, many forms of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, occur as a result of which the brain becomes vulnerable to oxidative stress, ultimately resulting in neuronal death. An anionic water-soluble conjugated polyfluorene derivative poly(9,9-bis(6-sulfate hexyl) fluorene-alt-1,4-phenylene) sodium salt (P1) that binds Fe³⁺ proteins with very high selectivity and sensitivity is reported here. The photophysical properties of P1 were modified by the interaction with ferric heme-containing proteins cytochrome c (Cc), methemoglobin (MetHb), and hemin. P1 was found to be highly sensitive toward Fe³⁺ heme proteins as compared to nonmetalloproteins. We observed that the respective activities of ferric heme proteins were inhibited and proteins were unfolded, due to modification in their heme microenvironment in the presence of the polymer P1. The observations reported in this article provide the first example for the use of a water-soluble conjugated polymer in applications, such as (1) to detect small quantities of iron proteins in aqueous medium/physiological condition with the highest K(sv) values of 2.27 × 10⁸ M⁻¹ for Cc, 3.81 × 10⁷ M⁻¹ for MetHb, and 5.31 × 10⁷ M⁻¹ for hemin; (2) to study the physiological effects of heme metalloproteins; (3) to visualize the folding events in real time; and (4) the inhibition activity of metalloproteins can be selectively studied using a conjugated polymer based assay system rapidly without interference from nonmetalloproteins at biological pH. All this is achieved by generating optical events, taking advantage of the bright fluorescence of anionic polyfluorene P1 in this case, that can be observed and monitored by modification in the absorption and emission color in real time.