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 prope rties of the electrostatic complex formed between the sulfonated azo d ye congo red (CR) and the peptide acetyl-YAAAKAAAAKAAAAKA-amide (YAK12 3). In contrast to amyloid, this complex had good solubility. Aqueous solutions of mixtures of YAK123 and CR at varying molar ratios n(YAK12 3)/n(CR) = R were prepared and characterized by UV/vis, CD, fluorescen ce, Raman, and CE techniques. End points in spectroscopic titrations o f YAK123 into CR solutions identified two stoichiometries: YAX123CR2 and YAK123CR, designated as ''1-2'' and ''1-1'', respectively. When Y AK123 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 fluor escence 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 cha racteristic 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 hel ix 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;d ma suggested aggregation of the 1-2 complex. The other two bands had a n isosbestic point, and their behavior suggested the formation of an a ggregated 1-1 complex. CE measurements of these mixtures showed a sing le 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. W hen R much greater than 1, there was predominantly free YAK123. From t he spectroscopic data, the following equilibria were inferred. When R much less than 1, the 1-2 complex was in equilibrium With free CR. Whe n 0 < R < 0.5, self-assembly to the aggregate (1-2), occurred. As R ap proached 0.5, free CR was consumed and bound CR formed cross-links bet ween YAK123 molecules, leading to the multimer (1-2),(1-1),. As R appr oached 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 solut ion used in preparing polyion multilayer films.