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Investigation of Self-Assembly upon Formation of an Electrostatic Complex of Congo Red and a Helical Peptide Thomas M. Cooper* and Morley O. Stone Air Force Research Laboratory, AFRL/MLPJ, 3005 P Street Suite 1, Wright-Patterson Air Force Base, Ohio 45433 Received June 12, 1998. In Final Form: September 17, 1998 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-YAAAKAAAAKAAAAKAamide (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 sCsNd and sNdNs 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 multilayer films.
Introduction Congo red (CR) is an anionic, dichroic dye that readily forms complexes with numerous materials. CR has been shown to bind to amyloid deposits resulting from Alzheimer’s disease, forming a birefringent complex having a green polarization color.1-7 Amyloid has a β-sheet conformation and tends to have poor solubility, making study of complex formation with CR more difficult. CR, when complexed to poly(L-lysine), shows induced optical activity.8 Optical activity has also been observed when CR binds to dehydrogenases and kinases.9-11 CR has also been investigated as a component of an ion-complexed * Corresponding author. Phone: 937-255-3808 x3157. Fax: 937255-1128. E-mail:
[email protected]. (1) Benditt, E. P.; Eriksen, N.; Berglund, C. Proc. Natl. Acad. Sci. 1970, 66, 1044. (2) Glenner, G. G.; Eanes, E. D.; Page, D. L. J. Histochem. Cytochem. 1972, 20, 821. (3) Sajid, J.; Elhaddaoui, A.; Turrell, S. J. Mol. Struct. 1997, 408/ 409, 181. (4) Gupta-Bansal, R.; Brunden, K. R. J. Neurochem. 1998, 70, 292. (5) Watson, D. J.; Lander, A. D.; Selkoe, D. J. J. Biol. Chem. 1997, 272, 31817. (6) Ashburn, T. T.; Han, H.; McGuinness, B. F.; Lansbury, P. T., Jr. Chem. Biol. 1996, 3, 351. (7) Han, H.; Cho, C.; Lansbury, P. T. J. Am. Chem. Soc. 1996, 118, 4506. (8) Yamamoto, H.; Nakazawa, A.; Hayakawa, T. J. Polym. Sci.: Polym. Lett. Ed. 1983, 21, 131. (9) Edwards, R.; Woody, R. Biochem. Biophys. Res. Commun. 1977, 79, 470. (10) Edwards, R.; Woody, R. Biochemistry 1979, 18, 5197. (11) Edwards, R.; Woody, R. J. Phys. Chem. 1983, 87, 1329.
polymer gel.12 Several investigations describe the optical properties of spin-coated CR-doped poly(vinyl alcohol) films.13,14 In our laboratory, we have successfully prepared CR-containing polyion multilayer films by the spontaneous adsorption technique.15-19 Model systems developed for study of the amyloid-CR complex, including insulin and poly(L-lysine),20,21 can give insight into the structure of these multilayers. To gain insight into the structure of these films, and to learn structure-property relationships, we have been investigating the optical properties of dyes complexed with synthetic peptides. We have found that the peptide acetyl-YAAAKAAAAKAAAAKA-amide (designated as YAK123)22 formed a soluble electrostatic (12) Shibayama, M.; Ikkai, F.; Nomura, S. Macromol. Symp. 1995, 93, 277. (13) Egami, C. S. Y.; Sugihara, O.; Okamoto, N.; Fujimura, H.; Nakagawa, K. F. H. Appl. Phys. B: Lasers Opt. 1997, B64, 471. (14) Vorflusev, V. P.; Kitzerow, H. S.; Chigrinov, V. G. Appl. Phys. A: Mater. Sci. Process. 1997, A64, 615. (15) Cooper, T. M.; Campbell, A. L.; Noffsinger, C.; Gunther-Greer, J.; Crane, R. L.; Adams, W. W. MRS Proc. 1994, 351, 239. (16) Cooper, T. M.; Campbell, A. L.; Crane, R. L. Langmuir 1995, 11, 2713. (17) Ariga, K.; Lvov, Y.; Kunitake, T. J. Am. Chem. Soc. 1997, 119, 2224. (18) Ariga, K.; Onda, M.; Lvov, Y.; Kunitake, T. Chem. Lett. 1997, 25. (19) Yoo, D.; Lee, J.; Rubner, M. F. Mater. Res. Soc. Symp. Proc. 1996, 413, 395. (20) Klunk, W. E.; Pettegrew, J. W.; Abraham, D. J. J. Histochem. Cytochem. 1989, 37, 1273. (21) Pigorsch, E.; Elhaddaoui, A.; Turrell, S. J. Mol. Struct. 1995, 348, 61.
10.1021/la9806911 CCC: $15.00 © 1998 American Chemical Society Published on Web 10/24/1998
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Langmuir, Vol. 14, No. 23, 1998 6663 elution time (min) ) -2.87 + 0.21
M2/3 Z
where M is the molecular weight and Z is the charge. A series of solutions with varying nYAK123/nCR ) R were dissolved in 18MΩ water. The running buffer was 0.1 M phosphate buffer, pH 2.5 (Bio-Rad), placed at both the capillary inlet and outlet. A 60 s preinjection cycle filled the cartridge with running buffer. The sample was injected with a 20 psi‚s injection. The polarity was positive at the inlet and negative at the outlet. Following injection, the CE run was performed by setting the voltage to 15 kV, having a 15 min run time, monitoring the absorbance at 200 nm, and keeping the cartridge at room temperature.
Figure 1. Structures of congo red and YAK123. The peptide amino acid sequence contains the one-letter codes for amino acids: Y ) tyrosine, A ) alanine, and K ) lysine. Congo red has a charge ) -2, and YAK123 has a charge ) +3.
complex with CR (Figure 1). We collected UV/vis, circular dichroism (CD), fluoresence, Raman, and capillary electrophoresis (CE) measurements on mixtures of YAK123 and CR with varying molar ratios R ) nYAK123/nCR. Two stoichiometries will be discussed: YAK123*CR2, designated as “1-2”, and YAK123*CR, designated as “1-1”. By probing binding with several optical techniques, we have found equilibria between the peptide and the dye formed 1-2 and 1-1 complexes. The 1-2 and 1-1 complexes underwent self-assembly into aggregates whose composition varied with R. Also, YAK123 served as a template for guiding CR aggregation into a parallel (H) stacking arrangement. Experimental Section Materials. The peptide acetyl-YAAAKAAAAKAAAAKAamide was prepared to >95% purity by custom peptide synthesis (Syn-Pep Corporation). The letters correspond to the one-letter amino acid codes: Y ) tyrosine, A ) alanine, and K ) lysine. To eliminate end effects resulting from the zwitterionic character of the N- and C-termini, the N-terminus was acetylated and the C-terminus had an amide group. HPLC data supplied by the manufacturer showed one component in the peptide. Mass spectroscopy performed by the manufacturer gave a single major peak with the molecular weight of 1460.0. We verified the purity of the peptide by capillary electrophoresis, giving a single peak. Congo red (Aldrich) was used without purification. TLC (mobile phase was water/ethanol) of CR showed a single spot. YAK123 and CR were dissolved in 18 MΩ water prior to spectroscopic measurement. Peptide concentration was measured assuming (275 nm) ) 1450 M-1 cm-1.23 CR concentration was measured assuming (498 nm) ) 40 100 M-1 cm-1.8,24 Instrumentation. UV/vis spectra were obtained with a Perkin-Elmer Lambda-9 spectrophotometer. Fluorescence spectra were obtained with a Perkin-Elmer LS 50B spectrometer. CD spectra were obtained with a calibrated Jasco J720 spectropolarimeter.25 Helix content was measured from the ellipticity at 222 nm.22,26 Raman spectra resulting from excitation at 1064 nm were obtained using a Bruker spectrometer. CE measurements were obtained using a Bio-Rad BioFocus 2000 system fitted with a 25 cm × 25 µm cartridge. The instrument was calibrated with the Bio-Rad peptide calibration set. The calibration curve was fit to the Offord relationship27 (22) Marqusee, S.; Robbins, V. H.; Baldwin, R. L. Proc. Natl. Acad. Sci. 1989, 86, 5286. (23) Brandts, J.; Kaplan, L. Biochemistry 1973, 12, 2011. (24) Green, F. J. The Sigma-Aldrich Handbook of Stains, Dyes and Indicators; Aldrich Chemical Co., Inc.: Milwaukee, WI, 1990. (25) Chen, Y.; Yang, J. Anal. Lett. 1977, 10, 3350. (26) Holtzer, M. E.; Holtzer, A.; Skolnick, J. Macromolecules 1983, 16, 173. (27) Grossman, P. D.; Colburn, J. C. Capillary Electrophoresis: Theory and Practice; Academic Press: San Diego, CA, 1992; p 264.
Results and Discussion UV/vis spectra of several anionic dyes, including methyl orange, Ponceau S, Evans blue, Eriochrome black, and CR, in the presence of YAK123 were measured. Changes in the UV/vis spectrum providing evidence of complex formation were observed only with CR. Figure 2 shows UV/vis spectra of a mixture containing excess YAK123. The major optical transitions of CR are the ππ* band at 498 nm, another ππ* band at 350 nm, and underlying low-intensity nπ* bands associated with the azo group.28,29 Upon binding, the λmax blue-shifted from 498 to 490 nm, accompanied by hypochromism and the appearance of two broad absorption bands. There was an isosbestic point at 350 nm, showing the chromophore existed in two states: free and bound. Addition of a drop of 1 N NaOH to the solution caused the spectrum to revert to its original form, suggesting electrostatic interactions between the peptide and CR caused the observed changes in the UV/vis spectrum. No hypochromism or blue shifting was observed in the UV/vis spectrum of CR in the presence of lysine hydrochloride in the same range of concentrations as those for YAK123. This observation suggests the positively charged lysine side chains need to be placed in a specific orientation in order for recognition and binding to occur with CR. A similar hypochromism and blue shift has been observed in the CR-poly(L-lysine) complex dissolved in a neutral aqueous solution.8,21 Red shifting and hypochromism have been observed in the binding of CR with poly(L-lysine) at pH 11,21 insulin,20 poly(vinyl alcohol),30 and the peptide EAK16.31 Such changes may be attributed to formation of ordered dye aggregates. Measurement of molar absorptivity as a function of R suggested the formation of a 1-2 complex (Figure 3). This result contrasted with that for the CR-poly(L-lysine) system, where the binding stoichiometry was consistent with a 1-1 CR-lysine monomer complex.8 The effects of aggregation on absorption spectra have been described by Tinoco.32 The excited-state wave function of the aggregate is a linear combination of monomer wave functions. Nearest neighbor interactions have the most significant effects on the aggregate UV/vis spectrum. Head-to-tail stacking of dyes leads to a red shift in the absorption spectrum (J aggregates), while a parallel stacking arrangement leads to a blue shift (H aggregates).33 CR has been shown to form self-associated supramolecular ribbon-like structures in water.34,35 Evidence of aggregate formation is shown by the similarity (28) Fabian, J.; Hartmann, H. Light Absorption of Organic Colorants; Springer-Verlag: Berlin, 1980. (29) Griffiths, J. Colour and Constitution of Organic Molecules; Academic Press: London, 1976. (30) Chung, W. H. Nucl. Technol. 1994, 105, 457. (31) Cooper, T. M.; Cline, S. M.; Campbell, A. L.; Adams, W. W. Polym. Prepr. 1996, 37, 721. (32) Tinoco, I. Adv. Chem. Phys. 1962, 4, 113. (33) The Theory of the Photographic Process; James, T. H., Ed.; Macmillan Company: New York, 1966; pp 244-246.
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xn )
Figure 2. UV/vis spectra of free CR, CR in the presence of YAK123 in DI water (R ) 1.5), and CR in the presence of 0.7 M KCl. In the free CR and CR-YAK123 systems, [CR] ) 4.3 × 10-5 M. In the presence of salt, [CR] ) 2.8 × 10-5 M.
Figure 3. Plot of molar extinction coefficient at 498 nm versus R, obtained from UV/vis spectra.
of the CR UV/vis spectrum in the presence of high salt concentration, where hypochromism and blue shift also occurred (Figure 2). This suggests the chromophores remained aggregated at R > 0.5 with no changes in nearest neighbor interactions that would affect the transition energy. What is the aggregation state of unbound CR in water? Temperature jump studies have been done on the association and dissociation reactions of CR.36 From these studies, the equilibrium constant for dimerization is 5.6 × 103 M-1. A spectrophotometric investigation of aggregation of the CR analogue benzopurpurin 4B reveals the equilibrium constant for higher aggregate formation follows the relation
log Kn ) (n - 1) log K2 where n is the aggregation number.37 The mole fraction of an n-mer is given by38 (34) Stopa, B. K. L.; Piekarska, B.; Roterman, I.; Rybarska, J.; Skowronek, M. Biochimie 1997, 79, 23. (35) Roterman, I.; No, T.; Piekarska, B.; Kaszuba, J.; Pawlicki, R.; Rybarska, J.; Konieczny, L. J. Phys. Pharm. 1993, 44, 213. (36) Yasunaga, T.; Nishikawa, S. Bull. Chem. Soc. Jpn. 1972, 45, 1262. (37) Hida, M.; Yabe, A.; Murayama, H.; Hayashi, M. Bull. Chem. Soc. Jpn. 1968, 41, 1776. (38) Hamada, K.; Kubota, H.; Ichimura, A.; Iijima, T.; Amiya, S. Ber. Bunsen-Ges. Phys. Chem. 1985, 89, 859.
nKn-1Cn1 C0
where C1 is the monomer concentration and C0 is the concentration of all forms of the dye. The CR concentration range of the current experiments was ∼10-5 M. In this concentration range, the dye monomer content was calculated to be 92%. In the presence of YAK123, aggregation into dimers and higher aggregates became more favored. X-ray crystal structures of complexes of basic amino acids with azo dyes have been published.39 A common hydrogen-bonding pattern observed is the formation of chains of dye molecules having the hydrogen bonding motif C22(10).40 Such a motif is possible in CR aggregates, where the motif is composed of the interactions S-C4C3-C2-C1-N-H ‚‚‚O-H2‚‚‚O(S), where the first group of atoms is located on one CR, followed by a water bridge to an oxygen bound to a sulfur on a second CR. The two hydrogen bond donors are an amino nitrogen and a water bridge nitrogen. The two hydrogen bond acceptors are a bridge water oxygen and a sulfonate oxygen. This hydrogen-bonding pattern could produce ribbon-like arrangements or head-to-tail arrangements of the dye. Complex formation caused peptide conformation changes (Figure 4). From the backbone CD spectrum (Figures 4 and 5), unbound YAK123 had 36% helix content. The low helix content resulted from electrostatic repulsion of the three positively charged lys residues. Unbound YAK123 consisted of a helical region ∼5-6 residues (1-2 turns) in length, with the remaining residues in a coil conformation. In the presence of excess CR, the 1-2 complex predominated, giving 67% helix content and ∼10 residues (3 turns) with a helical conformation (Figure 5). The presence of an isosbestic point (Figure 4) gave evidence for two YAK123 conformational states: free and bound. Aggregates formed from the 1-1 complex had 55% helix content, which suggests a solution containing excess YAK123 contains free peptide as well as an aggregate of 1-1 and 1-2 complexes. Free CR had weak fluorescence (Figure 6). The excitation spectrum of free CR was identical to CR’s UV/vis spectrum. Fluorescence intensity increased in the presence of YAK123 (Figure 7), with no change in the emission maximum. Addition of a drop of 1.0 N NaOH to the solution caused the increased fluorescence intensity to disappear. In contrast to the UV/vis data, the end point of intensity increase suggested the formation of a 1-1 complex. The curve had an S shape with an inflection point appearing near R ) 0.5 and leveling off at R ) 1 while the curve obtained from UV/vis measurements leveled off at R ) 0.5 (Figure 3). The variation in fluorescence intensity resulted from changes in quantum yield, given by41
φe )
φ*k0e k0e +
∑ki
where φ* is the formation efficiency of the emitting state, k0e is the rate constant for emission, and ∑ki is the sum of all rate constants that deactivate the excited state. Salt (39) Ojala, W. H.; Sudbeck, E. A.; Lu, L. K.; Richardson, T. I.; Lovrien, R. E.; Gleason, W. B. J. Am. Chem. Soc. 1996, 118, 2131. (40) Bernstein, J.; Davis, R. E.; Shimoni, L.; Chang, N. Angew. Chem., Int. Ed. Engl. 1995, 34, 1555. (41) Turro, N. J. Modern Molecular Photochemistry; University Science Books: Sausalito, 1991; p 609.
Complex of Congo Red and a Helical Peptide
Figure 4. Plot of CD spectra of YAK123 in the presence of CR. [CR]/[YAK123] ) 1/R ) 0, 0.25, 0.51, 0.75, 1.01, 1.27, 1.52, 1.77, 2.03, and 2.28.
Langmuir, Vol. 14, No. 23, 1998 6665
Figure 7. Plot of fluorescence intensity at 625 nm versus R for a CR solution excited at 500 nm. The data were corrected for the inner filter effect and normalized for concentration.53 Table 1. Raman Data of the YAK123-CR Complex ν(free)a
ν(bound)
∆ν
assignment
1156.9 1375.1 1594.0
1154.6 1373.3 1592.9
-2.3 -1.8 -1.1
sCsNd sNdNs aromatic sCdCs
a
Vibrational frequencies in wavenumbers.
Figure 5. Plot of YAK123 helix content as a function of [CR]/ [YAK123] ) 1/R.
Figure 8. Plot of the vibration frequency of the sCsNd stretch band as a function of R.
Figure 6. Plot of fluorescence spectra of CR in the presence of YAK123. R ) 0, 0.17, 0.34, 0.51, 0.68, 0.86, 1.03, 1.20, 1.37, 1.54, 1.71, 1.88, 2.10, 2.23, and 2.40. The arrow shows an increase in fluorescence intensity with R. [CR] ) 1.30 × 10-5 M. Excitation wavelength ) 500 nm; spectrometer slits set at 10 nm/10 nm.
bridges formed between sulfonate groups on CR and lysine ammonium groups on YAK123 may decrease CR’s conformation fluctuations, decreasing the rate of radiationless decay and increasing quantum yield. The slight resonance enhancement upon excitation of CR at 1064 nm gave Raman spectra of the chromophore in the presence of YAK123. No bands characteristic of YAK123 were observed in the Raman spectra. The Raman experiment probed electronic ground-state vibrational modes that had resonance enhancement with CR ππ* excited states. Table 1 lists the main Raman bands observed in free and bound CR. From literature data,
sNdNs group bands lie in the region 1380-1440 cm-1 and those of the sCsNd group were around 1154 cm-1.42-44 The largest spectral shift upon binding was observed in the sCsNd stretch band and the sNdNs stretch band. Smaller frequency decreases were observed in the aromatic sCdCs stretch band. All the shifts were to a lower wavenumber. The spectral shifts were small compared to that observed in CR-amyloid complexes, where there were significant shifts in the phenyl ring mode and the azo mode frequencies.3 The small changes in frequency suggest the bound dye was solvated and did not interact closely with the peptide. From the UV/vis data, CR aggregated when bound to YAK123. The small frequency decreases reflect bond order decreases resulting from changes in dye conformation and formation of exciton states. The data of Figure 8 show the frequency shifts were proportional to R and had an end point consistent with the formation of a 1-1 complex. Upon complex formation, CR exhibited intense induced CD. Four bands were observed (Figure 9). Upon addition of a drop of 1 N NaOH, the induced CD disappeared, (42) Hacker, H. Spectrochim. Acta 1965, 21, 1989. (43) Bassignana, P.; Cogrossi, C. Tetrahedron 1964, 20, 2361. (44) Elhaddaoui, A.; Delacourte, A.; Turrell, S. J. Mol. Struct. 1993, 294, 115.
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Figure 9. Induced CD spectra versus R. R varied from 0.06 to 2.1. The variations in molar ellipticity and the ellipticity maxima with R are shown in Figures 10 and 11.
Figure 11. (A, top) Ellipticity maxima of bands I and II of CR’s induced dichroism as a function of R. (B, bottom) Ellipticity maxima of bands III and IV of CR’s induced dichroism as a function of R. Figure 10. Molar ellipticity of CR’s induced dichroism as a function of R.
suggesting the complex did not form when YAK123 had a neutral charge. There was an isosbestic point at 328 nm between bands I and II but no isosbestic point between bands III and IV. The sign of the Cotton effect reflected the handedness of the transition dipole moment vectors of the two CR chromophores located in chiral positions with respect to each other.45 When R > 1, bands I and II (315 and 361 nm) had a Cotton effect reflecting negative chirality, while bands III and IV (461 and 529 nm) had a Cotton effect reflecting positive chirality.45 Both couplets were localized symmetrically about the two UV/vis absorption bands of CR (340 and 490 nm). The molar ellipticities had significant variation with R (Figure 10). Bands II and III showed most variation in the range 0 < R < 0.5 and reached an end point at R ) 0.5. The intensity of band I smoothly increased to R ) 1, although there was a distinct inflection point near R ) 0.5. The ellipticity of band IV increased to a maximum at R near 0.5 and then decreased and leveled off at R ) 1. The ellipticity maxima were a function of R (Figure 11). Bands I and IV showed slight variation with the concentration ratio. Band II red-shifted and band III blueshifted as R varied from 0 to 1, although most of the shift occurred between 0 and 0.5. As R approached 0, only the 1-2 complex was present (Figure 5), with band II extrapolating to 28 800 cm-1 and band III extrapolating to 20 400 cm-1. We performed CE measurements on mixtures of CR and YAK123 at varying R. For all concentration ratios, (45) Harada, N.; Nakanishi, K. Circular Dichroic Spectroscopy: Exciton Coupling in Organic Stereochemistry; University Science Books: Mill Valley, 1983; p 6.
a single band appeared. The CE experiment detected one type of aggregate in a CR/YAK123 solution with a given composition, rather than a mixture of free and bound YAK123. The retention time was a function of R, varying from 7 min with excess YAK123 to ∼12 min with excess CR. We calculated M2/3/Z from the calibration curve. From a spectrophotometric titration, the pKa of the two basic sites on CR was determined to be 4.5. CR’s charge was therefore zero at the CE experiment’s pH, and all positive charges in the complex were localized on the peptide. By assuming Z of the complex to be an integral multiple of 3, we calculated the number of CR molecules bound to YAK123 for various values of R in the electrophoresis solution (Figure 12). Although our other experiments were performed at neutral pH, we gained insight into the complex formation from the CE data. When Z ) 3, the stoichiometry was 1 - n, with n being a function of R. When R , 1, the complex was 1-2. When R ) 1, the complex was 1-1. When R . 1, no complex formed and only free peptide was detected. We also calculated expected stoichiometries assuming Z ) 6, 9, and 12 (Figure 12). For example, when R ) 0.1 and Z ) 12, a 1-6 complex was predicted. When R .1, a 1-2.5 complex was predicted. These results were not consistent with our other data. In contrast, when Z ) 3, the calculated results fit with the other experimental data. The CE data imply no higher order aggregates were observed at pH 2.5 due to repulsion between YAK123 species. Our results are also supported by literature obsevations that binding of CR has been shown to significantly affect the electrophoretic mobility of proteins.46,47 (46) Rybarska, J.; Konieczny, L.; Piekarska, B.; Stopa, B.; Roterman, I. J. Physiol. Pharmacol. 1995, 46, 221. (47) Piekarska, B.; Skowronek, M.; Rybarska, J.; Stopa, B.; Roterman, I.; Konieczny, L. Biochimie 1996, 78, 183.
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Figure 14. Multiple equilibria inferred from the experimental results.
Figure 12. Analysis of CE data. Abscissa: Molar ratio of YAK123 to CR in the electrophoresis solution. Ordinate: Calculated ratio of the number of moles of bound CR per mole of YAK123 obtained from the retention time and the instrument calibration curve.
Figure 13. Relative proportions of free CR, CR bound to the 1-2 complex, and CR bound to the 1-1 complex obtained from induced CD data (Figure 10) as a function of R.
The induced CD data suggested self-assembly occurred. Band I (Figures 9 and 10) showed a sigmoidal increase in intensity with increasing R, suggesting a cooperative transition. We analyzed the data from Figure 10 and obtained expressions for the mole fractions of the individual components according to the following expressions I [θ]I ) x1-1[θ] 1-1
(1)
IV IV [θ]IV ) x1-2[θ] 1-2 + x1-1[θ] 1-1
(2)
xCR + x1-2 + x1-1 ) 1
(3)
where x is the mole fraction of either free CR or CR bound in a 1-2 or 1-1 complex. The quantity [θ] is the molar ellipticity per mole of CR for bands I and IV. Bands II and III were not used in the analysis, as their ellipticity maxima had significant variation with R and lacked isosbestic points. Equation 1 describes the formation of aggregates containing 1-1 complexes, and eq 2 contains contributions from both 1-1 and 1-2 complexes. Equation 3 is a mass balance relation. When R > 1, we assumed I IV and [θ] 1-1 were estimated to be 16 700 x1-1 ) 1 and [θ] 1-1 2 dmol-1, respectively. From eq 1, x and 6500 deg‚cm 1-1 was calculated. Inserting these values into eq 2, the IV was calculated. To calculate xCR and product x1-2[θ] 1-2 IV x1-2, [θ] 1-2 was assumed to be 20 000 deg‚cm2 dmol-1. Figure 13 gives the three mole fractions calculated from induced CD data.
From these results multiple equilibria were inferred, one involving addition of two CR molecules to a positively charged complex and another involving addition of one YAK123 molecule to a negatively charged complex (Figure 14). Equilibrium I describes the formation of a 1-2 complex. Equilibrium II describes the observed aggregation of 1-2 complexes. Equilibrium III describes addition of YAK123 to an aggregate containing a mixture of 1-2 and 1-1 complexes. When R , 1, the data of Figures 5 and 13 show equilibrium I predominated. The 1-2 complex formed had a charge equaling -1. In the range 0 < R < 0.5, the data of Figure 11 give evidence that individual 1-2 complexes formed higher aggregates (12)m according to equilibrium II. The salt effect shown in Figure 2 gives evidence for dye aggregation occurring along with peptide aggregation. All the free CR was bound to YAK123 when R ) 0.5. In the range 0.5 < R < 1.0, aggregates containing both 1-2 and 1-1 complexes appeared as addition of YAK123 caused bound CR molecules to form cross-links between adjacent YAK123 molecules (equilibrium III). The 1-1 complex had a charge equaling +1. Evidence for conversion of aggregates containing mostly 1-2 complexes to those containing mostly 1-1 complexes was obtained from fluorescence, Raman, and induced CD data. The fluorescence data (Figure 7) showed a smooth increase in intensity in the range 0 < R < 1, leveling off when R > 1. The Raman data show shifts in the vibrational frequencies that are a function of R, leveling off at R ) 1 (Figure 8). Band I of the CD spectrum (Figure 10) also showed behavior mirroring that seen in the fluorescence and Raman data. In all three data sets, the midpoint of the curve appeared near R ) 0.5. The sigmoidal binding curves seen in Figures 7 and 10 suggested a cooperative transition to form the (1-1)n aggregate. According to the results of Figure 13, there was a roughly equal proportion of 1-1 and 1-2 complexes at the midpoint of the cooperative transition. The charge Q of the aggregate (1-2)m(1-1)n is Q ) n m. When R < 0.5, Q < 0, and when R > 0.5, Q > 0. At the midpoint, m ) n and the aggregate was electrically neutral. As R approached 1, m approached 0. When R > 1, n reached some equilibrium value and the solution contained increasing amounts of free YAK123. The CE data suggested that when R . 1, the solution primarily contained free YAK123. There have been several investigations of the electrostatic complex between poly(Llysine) and CR.8,21 Induced CD spectra of CR in the presence of poly(L-lysine) appear to have an isosbestic point, suggesting simpler equilibria than observed in this work.8 YAK123’s amino acid sequence has a mean hydropobicity 〈H〉 ) 0.09 and a mean helical hydrophobic moment 〈µH〉 ) -0.14, similar to that of helical segments of globular proteins.48 The low mean hydrophobicity and helical moment of this amino acid sequence gave good water solubility by projecting polar groups all around the helix and yet retaining some helical conformation. This also ensured the formation of a soluble dye-peptide complex. In our experiments, we prepared aqueous solutions (48) Eisenberg, D.; Weiss, R. M.; Terwilliger, T. C. Nature 1982, 299, 371.
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containing varying ratios of CR and YAK123. In all cases, the complex remained in solution. We did an earlier study on complexes between the peptide EAK16 (acetyl-AEAEAKAKAEAEAKAK-amide) and various dyes.31 In that instance, the complex tended to precipitate out of solution. EAK16 has a β-strand conformation insensitive to pH, temperature, and dye binding.31,49,50 The polyion-CR system is a ternary system consisting of peptide, CR, and water. The difference in solubility behavior might result from the ability of YAK123 to undergo changes in helix content upon binding. When a ligand binds to the peptide, minimization of the free energy of the solution can occur through conformation change, reorganization of the solvent shell around the complex, or formation of a precipitate. The rigidity of EAK16 prevents lowering of free energy by conformation change, so precipitation occurs. A high-resolution X-ray structure of CR reveals the considerable conformational flexibility of the biphenyl spacer, allowing for the hydrophobic regions of the molecule to fit into hydrophobic binding sites.51 The sulfonate groups orient anti with respect to the molecular axis and have an eclipsed conformation with respect to the naphthalene ring. Two biphenyl conformations were observed in the unit cell, one in which the biphenyl assumes a twisted (25°) conformation and one in which the biphenyl group is planar. The results demonstrate considerable biphenyl torsional flexibility, allowing for a wide range of conformations when CR binds to YAK123. The three-dimensional structure of a CR-porcine insulin complex has been determined, showing a single dye molecule intercalates between two globular insulins at an interface formed by two antiparallel β-strands.52 In contrast to the X-ray results, the sulfonate groups have a syn orientation with respect to the molecular axis. The sensitivity of orientation to environment results from the low torsional barrier between the biphenyls. When bound to YAK123, the CR sulfonate groups could be in either a syn or anti orientation. CR derivatives have been shown to have varying affinities for brain amyloid.7 By constraining the CR conformation to syn or anti, it would be possible to determine the importance of chromophore flexibility in binding to the peptide. The behavior of the aggregates observed in this work has similarities to examples of electrostatic complexes described in the literature. Complexes between sodium poly(styrenesulfonate) and diallyldimethylammonium chloride-acrylamide copolymers have viscosities that vary with the ratio of polycationic to polyanionic groups. The (49) Zhang, S.; Holmes, T.; Lockshin, C.; Rich, A. Proc. Natl. Acad. Sci. 1993, 90, 3334. (50) Zhang, S.; Lockshin, C.; Cook, R.; Rich, A. Biopolymers 1994, 34, 663. (51) Ojala, W. H.; Ojala, C. R.; Gleason, W. B. Antiviral Chem. Chemother. 1995, 6, 25. (52) Turnell, W. G.; Finch, J. T. J. Mol. Biol. 1992, 227, 1205.
Cooper and Stone
complexes have the minimum viscosity at the 1:1 stoichiometry.54 The turbidity of mixtures of colloidal SiO2 particles and poly(diallyldimethylammonium chloride) changes with the SiO2/polymer ratio. The solution of aggregates has the largest turbidity when the SiO2/ polymer particles have a net neutral charge.55 The ethidium bromide/closed circular duplex DNA complex changes its sedimentation rate with increasing dye/ nucleotide ratio.56 Intercalation between the base pairs unwinds the double helix, causing a lowered sedimentaton rate. When the superhelix is completely relaxed, the complex has the lowest sedimentation velocity. Addition of more ethidium bromide causes supercoiling in the opposite direction, increasing the sedimentation velocity. In the current work the aggregates formed of the 1-2 complex had a negative charge, while those formed from the 1-1 complex had a positive charge. Near R ) 0.5, cross-linking of CR between YAK123 molecules will lead to neutral aggregates containing both 1-2 and 1-1 complexes. Conclusions In this article we have presented spectroscopic evidence that the helical peptide YAK123 caused CR to undergo parallel stacking to form H aggregates. In contrast, the peptide EAK16 causes CR to undergo end-to-end stacking to form J aggregates.31 These two observations imply that peptides can be used as templates for controlling the structure of a dye aggregate or as a seed for growth of large single crystals. The technique of spontaneous adsorption is an approach for preparing polyion multilayer thin films containing these aggregates. It has been shown that the components of dipping solutions containing positively charged dyes and proteins premixed with linear polyions can be adsorbed onto a glass substrate.18 In this work we have shown that the characteristics of a peptidedye mixture are a strong function of composition. A mixture of YAK123 and CR will form complexes whose charges are a function of R. For example, when deposition of a monolayer with a negative charge onto a positively charged surface is required, a dipping solution with R < 0.5 should be prepared. The properties of polyion multilayer thin films produced from these mixtures will be described in a later publication. Acknowledgment. The authors thank Mr. Nathan Grebasch for collecting Raman spectra, Ms. Lori Halsey for CE measurements, and the Materials Directorate Lab Director’s Fund for support of this project. LA9806911 (53) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983. (54) Brand, F.; Dautzenberg, H. Langmuir 1997, 13, 2905. (55) Lvov, Y.; Ariga, K.; Onda, M.; Ichinose, I.; Kunitake, T. Langmuir 1997, 13, 6195. (56) Bauer, W. R.; Crick, F. H. C.; White, J. H. Sci. Am. 1980, 243, 129.
