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: YAX123*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 ma;dma 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 much less than 1, a 1-2 complex was identified. when R = 1, a 1-1 complex was observed. When R much greater than 1, there was predominantly free YAK123. From the spectroscopic data, the following equilibria were inferred. When R much less than 1, the 1-2 complex was in equilibrium With free CR. When 0 < R < 0.5, self-assembly to the aggregate (1-2), 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),(1-1),. As R approached 1, m approached 0 and the self-assembled complex became (1-1),. When R much greater than 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 multilayer films.