Investigation of Self-Assembly upon Formation of an Electrostatic Complex of Congo Red and a Helical Peptide

We have performed a detailed investigation of the solution-phase properties of the electrostatic complex formed between the sulfonated azo dye congo red (CR) and the peptide acetyl-YAAAKAAAAKAAAAKA-amide (YAK123). In contrast to amyloid, this complex had good solubility. Aqueous solutions of mixtures of YAK123 and CR at varying molar ratios n(YAK123)/n(CR) = R were prepared and characterized by UV/vis, CD, fluorescence, Raman, and CE techniques. End points in spectroscopic titrations of YAK123 into CR solutions identified two stoichiometries: YAK123*CR2 and YAK123*CR, designated as “1−2” and “1−1”, respectively. When YAK123 was added to a CR solution, the UV/vis spectrum of CR underwent hypochromism and a blue shift characteristic of H aggregate formation. The UV/vis end point identified a 1−2 complex. In contrast, the fluorescence intensity of CR increased to an end point characteristic of a 1−1 complex. The Raman spectrum of CR had small decreases in the CN and NN stretch frequencies with increasing R with an end point characteristic of a 1−1 complex. From the CD spectrum of YAK123, the end point of the titration identified a 1−2 complex and an increase in helix content from 36% to 67% was observed. The induced CD spectrum of CR in the presence of YAK123 had four bands whose variation with R gave evidence for 1−2 and 1−1 complexes. When R < 0.5, two of the CD bands lacked an isosbestic point and the variation in their ellipticity maxima suggested aggregation of the 1−2 complex. The other two bands had an isosbestic point, and their behavior suggested the formation of an aggregated 1−1 complex. CE measurements of these mixtures showed a single band whose retention time varied with R. When R ≪ 1, a 1−2 complex was identified. When R = 1, a 1−1 complex was observed. When R ≫ 1, there was predominantly free YAK123. From the spectroscopic data, the following equilibria were inferred. When R ≪ 1, the 1−2 complex was in equilibrium with free CR. When 0 < R < 0.5, self-assembly to the aggregate (1−2) m occurred. As R approached 0.5, free CR was consumed and bound CR formed cross-links between YAK123 molecules, leading to the multimer (1−2) m (1−1) n . As R approached 1, m approached 0 and the self-assembled complex became (1−1) n . When R ≫ 1, there was mostly free YAK123. The results suggest peptides can be used as templates for dye aggregation, seeds for growth of large single crystals, or a component in a dipping solution used in preparing polyion mult