VOL. 15, 2219-2226 (1976)
BIOPOLYMERS
Specificity in the Interaction Between Poly(Lglutamate) and Iron(II1) Complex Ions in Aqueous Solution M. BRANCA, M. E. MARINI, and B. PISPISA,* I s t i t u t o d i Chimica Fisica. Universitci d i R o m a , R o m e , I t a l y Synopsis The binding process between sodium poly(L-glutamate) and trans-2,2’,2”,2”’-tetrapyridyl-Fe(II1) complex ions in aqueous solution a t pH around 7 has been studied by means of equilibrium dialysis and optical measurements. The binding isotherm indicates the occurrence of a cooperative process, whereby bound molecules facilitate the association of additional molecules. According to circular dichroism (CD) data, this effect is coupled with that which sees a conformational change in the charged polypeptide upon progessive binding of complex counterions. All these features are discussed in the light of the structural characteristics of the interacting species. A stereochemical model of the association “complex” is proposed.
INTRODUCTION Association complexes between transition metal compounds and polyelectrolytes in solution have been studied in order to elucidate binding processes which often involve forces other than electrostatic ones,l-j and to investigate model systems similar to naturally occurring materials.6-’3 111) complex We have found that addition of 2,2’,2”,2”’-tetrapyridyliron( ions (Fig. 1) into sodium poly(L-glutamate) (Glu), solutions gives rise to a conformational changes in the charged polypeptide. A marked increase of the a-helical structure was observed a t those pH values where the coiled form normally predominates. Binding and optical data, as well as stereochemical considerations, suggest that such an effect primarily arises from a preferential association of the complex counterions to the helical conformation of the polymer.
EXPERIMENTAL The pseudo-octahedral trans iron(II1) derivative was obtained as already described.14 At the pH’s and concentrations with which we are primarily concerned, it is almost entirely in the mononuclear form: [Fe(tepy) (OH)z]+ (Fig. l).14 Poly(L-glutamic acid) (Miles-Yeda, M , 30,000) was * To whom correspondence should be addressed. 2219
8 1976 by John Wiley & Sons, Inc.
2220
BRANCA, MARINI, AND PISPISA
Y Fig. 1. Molecular model of trans-[Fe(tepy) (OH)z]+ complex ions.
converted to the sodium salt by NaOH 0.01 N . The stock solution was then exhaustively dialyzed against water to eliminate excess sodium ions. Concentrations of the polymer, C,, were determined by uv absorption at 200 nm ( t = 5,500). The final concentration was around 7 X loe4 M , referred to as a monomeric unit. In the alkaline pH region, Tris buffer (Sigma Chemical Corporation, US.) 0.01 M was employed. On the basis of preliminary optical measurements, interaction between the polymer or complex ions and buffer was ruled out. In the acidic region, the pH's of the polymer and'complex solutions were adjusted by adding appropriate amounts of standard acid or base. All measurements were performed on freshly prepared solutions, using doubly distilled water (conductivity less than 2.5 X loe6 ohm-l.crn-l, 20°C). Binding isotherms were obtained through equilibrium dialysis experiments a t 8' f 0.2"C (0.01M Tris buffer, pH = 7.2). The equilibrium was attained within 48 hr, with magnetic stirring of the external solutions containing the complex a t different concentrations. Before use, dialysis tubings (Thomas, US.)were carefully purified in the conventional way.15 A t the end of each set of experiments, the concentration of the complex in the external (polymer-free) solution was determined by uv absorption a t A,, 210 nm (emax 42,000), against a reference containing equimolar amounts of buffer. The concentration of the complex in the internal (polymer-containing) solution was determined at 300 nm, using previously plotted calibration curves. The concentration of bound counterions, Cb, was calculated as the difference between the concentration inside and outside the dialysis bag, that in the external solution being the free-complex concentration, Cf. The reproducibility was found to be better than f 6%. According to blank experiments; errors are not ascribable to retention of complex ions by membranes nor to polymer leakage. CD spectra were recorded on a Cary 61 instrument with quartz cells of 2, 1, or 0.1-cm path length; absorption spectra were determined on a Beckman DK-2A spectrophotometer and appropriate quartz cells. The stray light of the instrument was found to be practically zero a t 190 nm. The pH measurements were made with a Radiometer 26 pH meter with the use of semimicroelectrodes.
POLY(L-GLUTAMATE) AND IRON(II1) IONS
190
200
210
220
2221
230 240
X (nm/ Fig. 2. Conformational change in poly(i-glutamate) as determined by the variation of its CD spectral patterns with increased complex-to-polymer molar ratio ( C P ) , at pH = 7. Curve 2: (C/P) = 0.065 Curve 3: 0.100 Curve 4: 0.155 Curve 5: 0.200. Curve 1 refers to (Glu), solution a t the same pH, without added complex ions. Polymer concentration 7.55 X M ; optical path length of 1 cm.
RESULTS AND DISCUSSION A typical example of the “renaturating” effect exerted by [Fe(tepy) (OH)2]+complex ions on poly(L-glutamate) in aqueous solution is shown in Figure 2. As may be seen, the 215-217 ( R > 0) and 196 nm ( R < 0) CD bands, typical of the random coi1,16disappear, whereas the 220 and 208-210 nm ( R < 0) CD bands, typical of the a-helix, appear and strongly increase depending upon the amount of added complex ions. In Figure 3, the CD data at 220 nm of complex-polypeptide solutions are reported either as a function of pH at fixed complex-to-polymer molar ratios (C/P) or as a function of ( C P ) ratio at fixed pH. At pH 7, higher ( C P ) ratios than those reported in Figure 3 were avoided since the solutions show some opalescence which affects reproducibility of the optical data. By inspection of the figure, it appears that in both cases the curves exhibit an S shape, clearly reflecting the occurrence of a conformational transition in the polypeptide. Furthermore, the higher the complex-to-polymer ratio, the more the pH region of the helix-coil transformation is shifted upward. A stabilization effect on the helical structure of the substrate is observed even at a (C/P) ratio as low as 0.05. All these features suggest the occurrence of a specific binding site where forces other than electrostatic ones very likely play an important role. It is worth noting that the curves in Figure 3 level off in the presence of the complex ions at values of ellipticity lower than that of helical-(Glu),free complex solutions. This may be ascribed to a perturbation in the corresponding transitions of the polymer, due to the association, or to the
2222
BRANCA, MARINI, AND PISPISA
Fig. 3. Variation of ellipticity at 220 nm of polypeptide solutions in the presence of complex ions as a function of pH (unbroken lines) a t fixed complex-to-polymer molar ratios (Curve b (C/P) = 0.05; Curve c: 0.10), and as a function of (C/P) ratio (broken line) a t fixed pH (Curve d, pH = 7.0). The filled-in symbols and Curve a refer to polypeptide solutions without M. All measurements added iron(II1) derivative. Polymer concentration around 7.5 X were normalized for an optical path length of 1cm.
presence of an induced CD band of opposite sign in the bound molecules within the same frequency region, or to both effects. The CD spectral patterns reported in Figure 4, which refer to solutions a t pH=6, support the latter hypothesis. The ellipticity of the polypeptide is seen to decrease by some 17%as the (C/P) ratio increases from 0.05 to 0.10. On the other hand, at the given pH the a-helical conformation predominates, and hence the binding-induced “renaturation” effect is of minor importance. It may be concluded, therefore, that the bound complex molecules have an induced positive dichroic band also in the 200-220-nm wavelength region which subtracts rotational strength to the negative CD band of helical (Glu), a t 220 nm (Curves a-c of Fig. 3). Unfortunately, we could not investigate this effect a t higher (CD) ratios because precipitation phenomena were observed. On the other hand, a t fixed pH values higher than 6, such an effect is partially obscured by the conformational transition from the coiled form to the a-helical structure upon progressive binding of complex molecules (Curve d of Fig. 3). The marked difference between the CD curves of (Glu),-complex systems and those of (Glu),-free complex solutions a t the same value of pH as well as the characteristic shape of these curves (Fig. 2) strongly suggest that: 1) the formation of the helical structure is the predominant phenomenon that accompanies the interaction between the charged polypeptide and [Fe(tepy) (OH)z]+complex ions; and 2) the a-helical fraction of the substrate increases as the degree of association rises. In connection with the data reported in Figure 4, it must be pointed out that the CD bands recorded above 250 nm originate solely from the electronic transitions of the bound complex molecules, which are dissymmetrically perturbed by the asymmetric substrate. In principle, the induction of optical activity may be achieved by different mechanisms, not mutually
POLY(L-GLUTAMATE) AND IRON(II1) IONS
200
300
LOO
X(nrn)
2223
500
Fig. 4. CD spectral patterns of complex-polypeptide solutions (pH = 6) a t a complex-topolymer molar ratio of 0.05 (broken line) and 0.10 (unbroken line). Polymer concentration 7.4 X M; optical path length of 1cm. The dichroic bands recorded above 250 nm originate solely from the electronic transitions of the bound unchiral complex molecules (see text). Insert: extrinsic CD spectrum of bound counterions at pH = 6 (Curve 1)as compared to that at pH = 7.8 (Curve 2).
exclusive. The results shown in Figure 5, where the ellipticity of the two major CD bands of the bound counterions in the near uv region are plotted against pH, are suggestive of the occurrence of conformationally induced Cotton effects,17 resulting from the coulombic coupling of the electronic excitations of the molecules ligated in a chiral arrangement to the orderly polymer.1s-20 In fact, the rotational strength of both bands decreases in absolute value as the pH increases, although the degree of association increases since the polymer becomes fully charged. Furthermore, the trend of the curves clearly reflects the helix-coil transformation of the polymeric substrate. That the rotational strength of the CD band around 290 nm ( R < 0) is still different from zero even at pH values higher than 8 could also indicate, however, the presence of configurationally induced Cotton eff e c t ~ These . ~ ~ Cotton effects could arise from the interaction of the symmetric complex molecules with the locally asymmetric environment of the a-carbon atom of the polymer. Therefore, the formation of a coordination bonding between the y-carboxylate anions of the side chains, acting as unidentate ligands, and the trans -iron(111) derivative may be reasonably taken into account. Such a combination is sterically allowed (see below)
2224
BRANCA, MARINI, AND PISPISA
\ '\
-0.01
Y 'Ii 0.06
I'
-0.02
-0.03 -0.04
0.02
Fig. 5. Variation of ellipticity of the induced CD bands of the bound complex ions at 373 nm ( A ) and 287 nm ( 0 )as a function of pH. Complex-to-polymermolar ratio of 0.10; polymer concentration around 7.4 X M. All measurements were normalized for an optical pathlength of 2 cm (right-hand ordinate) and of 1 cm (left-hand ordinate).
and fully compatible with the high values of the induced molar ellipticity exhibited by the bound molecules (insert of Fig. 4). Binding data provide further support to these conclusions. The binding isotherm (8OC) of trans-[Fe (tepy) (OH)2]+in 7.4 X 10-4M (Glu), solutions containing 0.01 M Tris buffer (pH = 7.2) is reported in Figure 6. By inspection of this figure, it appears that: 1) the affinity for the pseudo-octahedral complex ions by (Glu), is very high; and 2) for Cf 0, the curve is convex to the free-complex concentration ordinate, indicating a cooperative process,21whereby bound molecules facilitate the association of additional molecules. As far as point 1) is concerned, the complex cations may be attracted closely enough to the negatively charged polyions so that additional stabilizing interactions occur, such as hydrophobic ones between the nonpolar tetrapyridyl ligand and the side chains of the macromolecule. In that case, the stereochemical requirements for a more extensive and possibly strong interaction are optimized by a preferential binding to the helical conformation. Molecular models clearly indicate that an intimate contact is achieved when the equatorial plane of the ligated molecules lies parallel to the helical axis, a relatively large portion of substrate being thus engaged by each bound molecule. This arrangement would also favor the coordination of the carboxylate groups in the apical position of the trans complex ions, which is expected to depress further the electrostatic repulsion along the chain. The overall effect should be such that the a-helical structure is stabilized and the conformational equilibrium is shifted towards the orderly form of the polymer. This is indeed the case, as shown in Figures 2 and 3.
-
POLY(L-GLUTAMATE) AND IRON(II1) IONS
0
1.0
0.5
2225
1.5
cf .105(m~i/lI
M (Glu), solutions Fig. 6. Binding isotherm (8°C) of [Fe(tepy) (OH),]+ ions in 7.4 X containing 0.01 M Tris buffer (pH = 7.2). Cb is the concentration of bound complex ions and C/ that of free complex ions, both expressed in mol/l.
The proposed stereochemistry is consistent with that reported for a number of association complexes between planar molecule^,^^,^^-^^ including h a e m i ~and ~ , helical ~ ~ polypeptides. Moreover, it may also account for the cooperative character of the association process [point 2)], which must be strictly related with the finding that, a t fixed pH, (Glu), undergoes a coil-a-helix transition as the complex-to-polymer ratio increases (Curve d of Fig. 3). In fact, bound complex molecules facilitate the association of other molecules (Fig. 6) because they very likely bring about an increase of the a-helical fraction in the polyelectrolyte. Finally, it is worth anticipating that preliminary data on the catalase-like activity of the system under investigation show a remarkable difference with respect to the same activity exhibited by [Fe(tepy)(OH)2)]+in the absence of macroions. In the former case a second-order kinetic process is observed, while in the latter case, a third-order reaction, with a partial order of 2 with respect to H202, is found. These results suggest that, upon association to (Glu),, only one site of the complex ions is eventually available for coordinating the peroxide molecules in the catalytic step, in agreement with the aforementioned hypothesis. In conclusion, the whole results show the occurrence of a specific binding site between trans - [Fe(tepy) (OH)2] ions and poly(L-glutamate), where interactions of mixed nature characterize the mode of association. Specificity in such a process arises from the stereochemical features of the complex molecules suitable for an intimate contact with the substrate. This produces in turn a conformational transition in the polypeptide in terms of a marked increase of a-helical structure a t those p H values where the coil form normally predominates. Comparison of the reported results with those obtained using other Fe(II1) complex ions, having different configuration, will be shown elsewhere. +
The authors wish to thank Dr. M. Barteri for helpful discussions throughout the work.
2226
BRANCA, MARINI, AND PISPISA
References 1. Crescenzi V. & Pispisa B. (1968) J. Polym. Sci. 6,1093-1100. 2. Ascoli F., Branca M., Mancini C. & Pispisa B. (1972) J. Chem. SOC.,Faraday I 68, 1213-1226. 3. Ascoli F., Branca M., Mancini C. & Pispisa B. (1973) Biopolymers 12,2431-2434. 4. Barteri M., Branca M. & Pispisa B. (1974) J. Chem. SOC.,Dalton Trans., 543-550. 5. Barteri M., Branca M. & Pispisa B. (1974) Biopolymers 13,2161-2167. 6. Blauer G. (1961) Nature 189,396-397. 7. Blauer, G. (1964) Biochim. Biophys. Acta 79,547-562. 8. Blauer G. & Ehrenberg A. (1963) Acta Chem. Scand. 17,8-12. 9. Blauer G. & Alfassi Z. B. (1967) Biochim. Biophys. Acta 133,206-218. 10. Wang J. H. & Brinigar W. S. (1960) Proc. Nut. Acad. Sci. U.S. 46,958-961. 11. Wang J. H. (1970) Accounts Chem. Res. 3,90-97. 12. Stryer L. (1961) Biochim. Biophys. Acta 54,397-399. 13. King T. E., Yong F. C. & Takemori S. (1966) Biochem. Biophys. Res. Comrnun. 22, 658-663. 14. Branca M., Pispisa B. & Aurisicchio C. (1976) J. Chem. Soc., Dalton Trans., (in press). 15. McPhie P. (1971) Methods in Enzymology, vol. 22, Academic, New York, pp. 23-32. 16. Myer Y. P. (1969) Macromolecules 2,624-628, and references cited therein. 17. Stryer L. & Blout E. R. (1961) J. Amer. Chem. SOC.83,1411-1418. 18. Jackson K. & Mason S. F. (1971) Trans.Faraday SOC.67,966-989. 19. Eyring H., Liu H,-C. & Caldwell D. (1968) Chem. Reu. 68,525-540, and references cited therein. 20. Tinoco I., Jr. (1962) Aduan. Chem. Phys. 4,113-160. 21. Blake A. & Peacocke A. R. (1968) Biopolymers 6,1225-1253. 22. Ballard R. E., McCaffery A. J. & Mason S. F. (1966) Biopolymers 4,97-106. 23. Sato Y. & Hatano M. (1973) Bull. Chem. SOC.Jap. 46,3339-3344. 24. Hatano M., Yoneyama M. & Sat0 Y. (1973) Biopolymers 12,895-903.
Received January 26,1976 Returned for revision April 12,1976 Accepted May 19,1976
BIOPOLYMERS
Specificity in the Interaction Between Poly(Lglutamate) and Iron(II1) Complex Ions in Aqueous Solution M. BRANCA, M. E. MARINI, and B. PISPISA,* I s t i t u t o d i Chimica Fisica. Universitci d i R o m a , R o m e , I t a l y Synopsis The binding process between sodium poly(L-glutamate) and trans-2,2’,2”,2”’-tetrapyridyl-Fe(II1) complex ions in aqueous solution a t pH around 7 has been studied by means of equilibrium dialysis and optical measurements. The binding isotherm indicates the occurrence of a cooperative process, whereby bound molecules facilitate the association of additional molecules. According to circular dichroism (CD) data, this effect is coupled with that which sees a conformational change in the charged polypeptide upon progessive binding of complex counterions. All these features are discussed in the light of the structural characteristics of the interacting species. A stereochemical model of the association “complex” is proposed.
INTRODUCTION Association complexes between transition metal compounds and polyelectrolytes in solution have been studied in order to elucidate binding processes which often involve forces other than electrostatic ones,l-j and to investigate model systems similar to naturally occurring materials.6-’3 111) complex We have found that addition of 2,2’,2”,2”’-tetrapyridyliron( ions (Fig. 1) into sodium poly(L-glutamate) (Glu), solutions gives rise to a conformational changes in the charged polypeptide. A marked increase of the a-helical structure was observed a t those pH values where the coiled form normally predominates. Binding and optical data, as well as stereochemical considerations, suggest that such an effect primarily arises from a preferential association of the complex counterions to the helical conformation of the polymer.
EXPERIMENTAL The pseudo-octahedral trans iron(II1) derivative was obtained as already described.14 At the pH’s and concentrations with which we are primarily concerned, it is almost entirely in the mononuclear form: [Fe(tepy) (OH)z]+ (Fig. l).14 Poly(L-glutamic acid) (Miles-Yeda, M , 30,000) was * To whom correspondence should be addressed. 2219
8 1976 by John Wiley & Sons, Inc.
2220
BRANCA, MARINI, AND PISPISA
Y Fig. 1. Molecular model of trans-[Fe(tepy) (OH)z]+ complex ions.
converted to the sodium salt by NaOH 0.01 N . The stock solution was then exhaustively dialyzed against water to eliminate excess sodium ions. Concentrations of the polymer, C,, were determined by uv absorption at 200 nm ( t = 5,500). The final concentration was around 7 X loe4 M , referred to as a monomeric unit. In the alkaline pH region, Tris buffer (Sigma Chemical Corporation, US.) 0.01 M was employed. On the basis of preliminary optical measurements, interaction between the polymer or complex ions and buffer was ruled out. In the acidic region, the pH's of the polymer and'complex solutions were adjusted by adding appropriate amounts of standard acid or base. All measurements were performed on freshly prepared solutions, using doubly distilled water (conductivity less than 2.5 X loe6 ohm-l.crn-l, 20°C). Binding isotherms were obtained through equilibrium dialysis experiments a t 8' f 0.2"C (0.01M Tris buffer, pH = 7.2). The equilibrium was attained within 48 hr, with magnetic stirring of the external solutions containing the complex a t different concentrations. Before use, dialysis tubings (Thomas, US.)were carefully purified in the conventional way.15 A t the end of each set of experiments, the concentration of the complex in the external (polymer-free) solution was determined by uv absorption a t A,, 210 nm (emax 42,000), against a reference containing equimolar amounts of buffer. The concentration of the complex in the internal (polymer-containing) solution was determined at 300 nm, using previously plotted calibration curves. The concentration of bound counterions, Cb, was calculated as the difference between the concentration inside and outside the dialysis bag, that in the external solution being the free-complex concentration, Cf. The reproducibility was found to be better than f 6%. According to blank experiments; errors are not ascribable to retention of complex ions by membranes nor to polymer leakage. CD spectra were recorded on a Cary 61 instrument with quartz cells of 2, 1, or 0.1-cm path length; absorption spectra were determined on a Beckman DK-2A spectrophotometer and appropriate quartz cells. The stray light of the instrument was found to be practically zero a t 190 nm. The pH measurements were made with a Radiometer 26 pH meter with the use of semimicroelectrodes.
POLY(L-GLUTAMATE) AND IRON(II1) IONS
190
200
210
220
2221
230 240
X (nm/ Fig. 2. Conformational change in poly(i-glutamate) as determined by the variation of its CD spectral patterns with increased complex-to-polymer molar ratio ( C P ) , at pH = 7. Curve 2: (C/P) = 0.065 Curve 3: 0.100 Curve 4: 0.155 Curve 5: 0.200. Curve 1 refers to (Glu), solution a t the same pH, without added complex ions. Polymer concentration 7.55 X M ; optical path length of 1 cm.
RESULTS AND DISCUSSION A typical example of the “renaturating” effect exerted by [Fe(tepy) (OH)2]+complex ions on poly(L-glutamate) in aqueous solution is shown in Figure 2. As may be seen, the 215-217 ( R > 0) and 196 nm ( R < 0) CD bands, typical of the random coi1,16disappear, whereas the 220 and 208-210 nm ( R < 0) CD bands, typical of the a-helix, appear and strongly increase depending upon the amount of added complex ions. In Figure 3, the CD data at 220 nm of complex-polypeptide solutions are reported either as a function of pH at fixed complex-to-polymer molar ratios (C/P) or as a function of ( C P ) ratio at fixed pH. At pH 7, higher ( C P ) ratios than those reported in Figure 3 were avoided since the solutions show some opalescence which affects reproducibility of the optical data. By inspection of the figure, it appears that in both cases the curves exhibit an S shape, clearly reflecting the occurrence of a conformational transition in the polypeptide. Furthermore, the higher the complex-to-polymer ratio, the more the pH region of the helix-coil transformation is shifted upward. A stabilization effect on the helical structure of the substrate is observed even at a (C/P) ratio as low as 0.05. All these features suggest the occurrence of a specific binding site where forces other than electrostatic ones very likely play an important role. It is worth noting that the curves in Figure 3 level off in the presence of the complex ions at values of ellipticity lower than that of helical-(Glu),free complex solutions. This may be ascribed to a perturbation in the corresponding transitions of the polymer, due to the association, or to the
2222
BRANCA, MARINI, AND PISPISA
Fig. 3. Variation of ellipticity at 220 nm of polypeptide solutions in the presence of complex ions as a function of pH (unbroken lines) a t fixed complex-to-polymer molar ratios (Curve b (C/P) = 0.05; Curve c: 0.10), and as a function of (C/P) ratio (broken line) a t fixed pH (Curve d, pH = 7.0). The filled-in symbols and Curve a refer to polypeptide solutions without M. All measurements added iron(II1) derivative. Polymer concentration around 7.5 X were normalized for an optical path length of 1cm.
presence of an induced CD band of opposite sign in the bound molecules within the same frequency region, or to both effects. The CD spectral patterns reported in Figure 4, which refer to solutions a t pH=6, support the latter hypothesis. The ellipticity of the polypeptide is seen to decrease by some 17%as the (C/P) ratio increases from 0.05 to 0.10. On the other hand, at the given pH the a-helical conformation predominates, and hence the binding-induced “renaturation” effect is of minor importance. It may be concluded, therefore, that the bound complex molecules have an induced positive dichroic band also in the 200-220-nm wavelength region which subtracts rotational strength to the negative CD band of helical (Glu), a t 220 nm (Curves a-c of Fig. 3). Unfortunately, we could not investigate this effect a t higher (CD) ratios because precipitation phenomena were observed. On the other hand, a t fixed pH values higher than 6, such an effect is partially obscured by the conformational transition from the coiled form to the a-helical structure upon progressive binding of complex molecules (Curve d of Fig. 3). The marked difference between the CD curves of (Glu),-complex systems and those of (Glu),-free complex solutions a t the same value of pH as well as the characteristic shape of these curves (Fig. 2) strongly suggest that: 1) the formation of the helical structure is the predominant phenomenon that accompanies the interaction between the charged polypeptide and [Fe(tepy) (OH)z]+complex ions; and 2) the a-helical fraction of the substrate increases as the degree of association rises. In connection with the data reported in Figure 4, it must be pointed out that the CD bands recorded above 250 nm originate solely from the electronic transitions of the bound complex molecules, which are dissymmetrically perturbed by the asymmetric substrate. In principle, the induction of optical activity may be achieved by different mechanisms, not mutually
POLY(L-GLUTAMATE) AND IRON(II1) IONS
200
300
LOO
X(nrn)
2223
500
Fig. 4. CD spectral patterns of complex-polypeptide solutions (pH = 6) a t a complex-topolymer molar ratio of 0.05 (broken line) and 0.10 (unbroken line). Polymer concentration 7.4 X M; optical path length of 1cm. The dichroic bands recorded above 250 nm originate solely from the electronic transitions of the bound unchiral complex molecules (see text). Insert: extrinsic CD spectrum of bound counterions at pH = 6 (Curve 1)as compared to that at pH = 7.8 (Curve 2).
exclusive. The results shown in Figure 5, where the ellipticity of the two major CD bands of the bound counterions in the near uv region are plotted against pH, are suggestive of the occurrence of conformationally induced Cotton effects,17 resulting from the coulombic coupling of the electronic excitations of the molecules ligated in a chiral arrangement to the orderly polymer.1s-20 In fact, the rotational strength of both bands decreases in absolute value as the pH increases, although the degree of association increases since the polymer becomes fully charged. Furthermore, the trend of the curves clearly reflects the helix-coil transformation of the polymeric substrate. That the rotational strength of the CD band around 290 nm ( R < 0) is still different from zero even at pH values higher than 8 could also indicate, however, the presence of configurationally induced Cotton eff e c t ~ These . ~ ~ Cotton effects could arise from the interaction of the symmetric complex molecules with the locally asymmetric environment of the a-carbon atom of the polymer. Therefore, the formation of a coordination bonding between the y-carboxylate anions of the side chains, acting as unidentate ligands, and the trans -iron(111) derivative may be reasonably taken into account. Such a combination is sterically allowed (see below)
2224
BRANCA, MARINI, AND PISPISA
\ '\
-0.01
Y 'Ii 0.06
I'
-0.02
-0.03 -0.04
0.02
Fig. 5. Variation of ellipticity of the induced CD bands of the bound complex ions at 373 nm ( A ) and 287 nm ( 0 )as a function of pH. Complex-to-polymermolar ratio of 0.10; polymer concentration around 7.4 X M. All measurements were normalized for an optical pathlength of 2 cm (right-hand ordinate) and of 1 cm (left-hand ordinate).
and fully compatible with the high values of the induced molar ellipticity exhibited by the bound molecules (insert of Fig. 4). Binding data provide further support to these conclusions. The binding isotherm (8OC) of trans-[Fe (tepy) (OH)2]+in 7.4 X 10-4M (Glu), solutions containing 0.01 M Tris buffer (pH = 7.2) is reported in Figure 6. By inspection of this figure, it appears that: 1) the affinity for the pseudo-octahedral complex ions by (Glu), is very high; and 2) for Cf 0, the curve is convex to the free-complex concentration ordinate, indicating a cooperative process,21whereby bound molecules facilitate the association of additional molecules. As far as point 1) is concerned, the complex cations may be attracted closely enough to the negatively charged polyions so that additional stabilizing interactions occur, such as hydrophobic ones between the nonpolar tetrapyridyl ligand and the side chains of the macromolecule. In that case, the stereochemical requirements for a more extensive and possibly strong interaction are optimized by a preferential binding to the helical conformation. Molecular models clearly indicate that an intimate contact is achieved when the equatorial plane of the ligated molecules lies parallel to the helical axis, a relatively large portion of substrate being thus engaged by each bound molecule. This arrangement would also favor the coordination of the carboxylate groups in the apical position of the trans complex ions, which is expected to depress further the electrostatic repulsion along the chain. The overall effect should be such that the a-helical structure is stabilized and the conformational equilibrium is shifted towards the orderly form of the polymer. This is indeed the case, as shown in Figures 2 and 3.
-
POLY(L-GLUTAMATE) AND IRON(II1) IONS
0
1.0
0.5
2225
1.5
cf .105(m~i/lI
M (Glu), solutions Fig. 6. Binding isotherm (8°C) of [Fe(tepy) (OH),]+ ions in 7.4 X containing 0.01 M Tris buffer (pH = 7.2). Cb is the concentration of bound complex ions and C/ that of free complex ions, both expressed in mol/l.
The proposed stereochemistry is consistent with that reported for a number of association complexes between planar molecule^,^^,^^-^^ including h a e m i ~and ~ , helical ~ ~ polypeptides. Moreover, it may also account for the cooperative character of the association process [point 2)], which must be strictly related with the finding that, a t fixed pH, (Glu), undergoes a coil-a-helix transition as the complex-to-polymer ratio increases (Curve d of Fig. 3). In fact, bound complex molecules facilitate the association of other molecules (Fig. 6) because they very likely bring about an increase of the a-helical fraction in the polyelectrolyte. Finally, it is worth anticipating that preliminary data on the catalase-like activity of the system under investigation show a remarkable difference with respect to the same activity exhibited by [Fe(tepy)(OH)2)]+in the absence of macroions. In the former case a second-order kinetic process is observed, while in the latter case, a third-order reaction, with a partial order of 2 with respect to H202, is found. These results suggest that, upon association to (Glu),, only one site of the complex ions is eventually available for coordinating the peroxide molecules in the catalytic step, in agreement with the aforementioned hypothesis. In conclusion, the whole results show the occurrence of a specific binding site between trans - [Fe(tepy) (OH)2] ions and poly(L-glutamate), where interactions of mixed nature characterize the mode of association. Specificity in such a process arises from the stereochemical features of the complex molecules suitable for an intimate contact with the substrate. This produces in turn a conformational transition in the polypeptide in terms of a marked increase of a-helical structure a t those p H values where the coil form normally predominates. Comparison of the reported results with those obtained using other Fe(II1) complex ions, having different configuration, will be shown elsewhere. +
The authors wish to thank Dr. M. Barteri for helpful discussions throughout the work.
2226
BRANCA, MARINI, AND PISPISA
References 1. Crescenzi V. & Pispisa B. (1968) J. Polym. Sci. 6,1093-1100. 2. Ascoli F., Branca M., Mancini C. & Pispisa B. (1972) J. Chem. SOC.,Faraday I 68, 1213-1226. 3. Ascoli F., Branca M., Mancini C. & Pispisa B. (1973) Biopolymers 12,2431-2434. 4. Barteri M., Branca M. & Pispisa B. (1974) J. Chem. SOC.,Dalton Trans., 543-550. 5. Barteri M., Branca M. & Pispisa B. (1974) Biopolymers 13,2161-2167. 6. Blauer G. (1961) Nature 189,396-397. 7. Blauer, G. (1964) Biochim. Biophys. Acta 79,547-562. 8. Blauer G. & Ehrenberg A. (1963) Acta Chem. Scand. 17,8-12. 9. Blauer G. & Alfassi Z. B. (1967) Biochim. Biophys. Acta 133,206-218. 10. Wang J. H. & Brinigar W. S. (1960) Proc. Nut. Acad. Sci. U.S. 46,958-961. 11. Wang J. H. (1970) Accounts Chem. Res. 3,90-97. 12. Stryer L. (1961) Biochim. Biophys. Acta 54,397-399. 13. King T. E., Yong F. C. & Takemori S. (1966) Biochem. Biophys. Res. Comrnun. 22, 658-663. 14. Branca M., Pispisa B. & Aurisicchio C. (1976) J. Chem. Soc., Dalton Trans., (in press). 15. McPhie P. (1971) Methods in Enzymology, vol. 22, Academic, New York, pp. 23-32. 16. Myer Y. P. (1969) Macromolecules 2,624-628, and references cited therein. 17. Stryer L. & Blout E. R. (1961) J. Amer. Chem. SOC.83,1411-1418. 18. Jackson K. & Mason S. F. (1971) Trans.Faraday SOC.67,966-989. 19. Eyring H., Liu H,-C. & Caldwell D. (1968) Chem. Reu. 68,525-540, and references cited therein. 20. Tinoco I., Jr. (1962) Aduan. Chem. Phys. 4,113-160. 21. Blake A. & Peacocke A. R. (1968) Biopolymers 6,1225-1253. 22. Ballard R. E., McCaffery A. J. & Mason S. F. (1966) Biopolymers 4,97-106. 23. Sato Y. & Hatano M. (1973) Bull. Chem. SOC.Jap. 46,3339-3344. 24. Hatano M., Yoneyama M. & Sat0 Y. (1973) Biopolymers 12,895-903.
Received January 26,1976 Returned for revision April 12,1976 Accepted May 19,1976
