Circular dichroism and magnetic circular dichroism of the haemin-poly(l -lysine) complex system

Circular dichroism and magnetic circular dichroism of the haemin-poly(t-lysine) complex system Seigo Yamamoto, Tsunenori Nozawa and Masahiro Hatano Chemical Research Institute of Non-Aqueous Solutions, Tohoku University, Sendai, Japan (Received 20 June 1973; revised 2 January 1974) Circular dichroism (c.d.) and magnetic circular dichroism (m.c.d.) of the haemin-poly(Llysine) (PLL) complex system in water were examined in the Soret and O region with variation of pH. C.d. bands were induced in the Soret region and their magnitudes rose with increase in the helix content of PLL. M.c.d. also showed a nearly similar trend. At pH 10.7 where PLL has the s-helix conformation, one negative and one positive c.d. band centred at around 25.5 and 24.0x 103cm-1 respectively, were observed in the Soret region. The wavenumbers of these two c.d. bands nearly correspond with each of the m.c.d. bands in this region. At pH 8.7 where PLL has a random coil form, we could not detect any c.d. band in the Soret region. The m.c.d, spectrum was very small at pH 8.7 in comparison with that at pH 10-7. The m.c.d, in the O region indicates that the haemin in its PLL complex exists in both a low spin and a high spin form at pH 10-7. From these experimental results, the m.c.d, character of the bound haemin as well as its electronic structure are discussed.

INTRODUCTION Since haemin-poly(L-lysine) (PLL) complexes are considered as a model for haemichromes of biological origin, the haemin-PLL complexes have previously been investigated by measurements of absorption spectrum, optical rotatory dispersion and paramagnetic properties 1-5. Blauer and his collaborators prepared the PLL complex and investigated its nature extensively, on the basis of the absorption spectra and the paramagnetic properties 1-5. The Cotton effect of the haemin- . PLL complex was observed and related to the helix structure of PLL by Stryer6. As a similar system, the poly(L-histidine)-haemin system was studied with circular dichroism (c.d.) by Beychok7. Although the structure of the haemin-PLL complex has been partly clarified by Blauer et al. l-a, the electronic states of bound haemin have not been fully discussed yet. Since magnetic circular dichroism (m.c.d.) has been found to be one of the most sensitive optical techniques for studying the electronic state s, we investigated the m.c.d, of the haemin-PLL with its c.d., and both its structure and electronic state are discussed. EXPERIMENTAL Materials Poly(L-lysine.HBr) (PLL.HBr) was prepared by the conventional method a. An average degree of polymerization (DP) of this sample was 920. Ferrihaeme chloride (haemin) was obtained from Daiichi Pure Chemicals Co. Ltd.

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Complex formation The haemin-PLL complexes were prepared by Blauer's method 3. The pH of the mixed solution was brought to the desired pH by addition of 0.1 N NaOH or 0.1 N HC1. The final lysyl residue concentration of PLL in the solution was 5.4 × 10-3 M, and that of haemin was 3-6 x 10-SM. The lysyl residue to haemin molar ratio was kept at 150. Measurements The c.d. and m.c.d, measurements were carried out with a Jasco Model J-20A spectropolarimeter with or without a 12.5 × 103 G electric magnet. The intensity of c.d. and m.c.d, are expressed by the molar ellipticity [0] or [0]~r with the unit of degree cmZ/dmol or degree cm2/dmol G respectively. The haemin concentration is used for the calculation of [0] or [0]M except for [0] at 45"0 ×103 cm-1. The absorption spectra were measured with a Hitachi Model EPS-3T spectrometer. The pH measurements of the solutions were made with a Toa Denpa Kogyo Model HM-8 pH meter. The titration vessel was continuously purged with nitrogen during the pH titration in order to exclude the effect of CO2. All measurements were taken at r o o m temperature. RESULTS AND DISCUSSION

C.d. spectra of the haemin-PLL complex Figure 1 shows the c.d. spectra in the Soret region of the haemin-PLL complex in aqueous solution at

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Figure 3 Soret absorption spectra of haemin-PLL complex as a function of pH. The molar extinction coefficient e is calculated for the haemin molar concentration (H). (R/H=150; H = 3 . 6 x 10 5 M.) - - , pH 10.7; . . . . , pH 9'9; , pH 9'6; . . . . , pH 8"7

bands at 25.5 and 24.0x 103cm-1 and % helix of haemin-PLL

various p H values. At p H 10.7 there are two c.d. bands of opposite signs with the negative one at the lower wavenumber. The two c.d. bands centred at around 25.5 and 24.0x 103cm -1 become weak with decreasing p H value. At p H 8.7, the two c.d. bands were no longer detectable within experimental error. The magnitudes [0] of the two c.d. bands varied with the p H value as shown in Figure 2. The solid and open circles represent the variation in the magnitudes of the induced c.d. bands at 25.5 and 24.0x 10~cm -1 respectively. In this Figure, the open square represents the variation in the magnitude of the c.d. band at 4 5 . 0 x 1 0 3 c m -1 as a function of pH, which corresponds to the helix content of P L L in the system. The s-helix contents were calculated with the assumption that [0]=30 000 for 100 K helix 9 and are shown in Figure 2. It should be mentioned that PLL in the h a e m i n - P L L complex shows a helix-coil transition in the same p H range as that of P L L itself in aqueous solution 9. Figure 2 indicates that these two c.d. bands are induced only in the helix region of PLL. As is evident from the mechanism of helix-coil transition 9, PLL in the complex should keep the helix form in the p H region where the amino groups in the side chains deprotonate and coordinate to the haemin iron. Therefore, the induced c.d. should have its origin in the fixation of the haemin

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C.d. and m.c.d, of the haemin-poly(L-lysine) complex: S. Yamamoto et aL Absorption spectra of the haemin-PLL complex Figure 3 shows the absorption spectra of the haeminPLL complex at several pH values. The spectral feature is in accordance with that reported by Blauer et al. 1, 3. The absorption spectra at pH 10.7 show a typical haemichrome spectra 1, 3. The lack of isosbesiticity in Figure 3, especially at pH 9.6 can be attributed to the presence of some intermediate complex species, one of which is probably the haemin with one amino ligand. M.c.d. spectra of the haemin-PLL complex Figure 4 shows the m.c.d, spectra in the Soret region of the haemin-PLL complex. The magnitude of the m.c.d, band at pH 10.7 increased by a factor of 10--20 in comparison with those at pH 8.7. Since some haemichromes with nitrogen bases, such as pyridine and imidazole, exhibit m.c.d, with similar shape and magnitude in the Soret region (Figure 5), the large magnitudes of the m.c.d, bands centred at 24 and 25 x 103cm -1 are also indicative of the formation of a haemichrome as suggested by the absorption spectra. In the Q band region the haemin-PLL exhibited some rather complex m.c.d, spectra both at pH 10.7 and at pH 8.7 (Figures 6 and 7); however, it can be interpreted by comparison with the following m.c.d, of the pyridine-haemin complex. The pyridine-haemin complex showed a time variation in the m.c.d. (Figure 8). Curve (A) is the m.c.d, of the haemin in the absence of pyridine. In these conditions,

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3:!2 POLYMER, 1974, Vol 15, dune

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Blauer et al. reported that the haemin forms a high spin dimer 4. Hence, curve (A) should be a m.c.d, for a high spin type haemin. The m.c.d, long enough after an addition of pyridine is the typical low spin type m.c.d, for haemichrome with two pyridines. Therefore the m.c.d, changes with time from (A) to (D) is considered to correspond to haemichrome formation from the hemin dimer. From the fact that the m.c.d, for the haemin-PLL at pH 10.7 is similar to that for the haemin-pyridine complex (B), the m.c.d, in the Q region therefore suggests that the haemin-PLL complex at this pH has both high and low spin type haemins. Free haemin dimer and/or haemin with only one amino nitrogen may be the high spin type. The low spin type will correspond to the haemin bound to PLL with two amino nitrogens. When we compare the m.c.d for the free haemin (Figure 8) with that for the haemin complex with PLL at pH 8-7 (Figure 9) in the Q region, we find that they are similar to each other except in the high wavenumber region (18-22 x 103cm -1) where the charge transfer bands make a simple comparison difficult.

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Figure 8 ' '. ' 18 14 v x IO-3(cm -I ) Figure 6 Absorption ( ) and magnetic circular dichroism (. . . . ) and circular dichroism (. . . . ) spectra of haemin-PLL complex at pH 10.7. (R/H=150; H=3-6x10-SM)

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The haemin in the presence of PLL forms the 'green complex' at pH 8-71, ~. It has the effective magnetic moment/~eff of 2"7 Bohr magneton (! Bohn magneton = 9.273x 10-Z4Am2) which corresponds to a fairly low spin type haemin. Blauer and his coworkers attributed

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it to the formation of a micellar aggregate of the haemin itself. In the absence of PLL the haemin has Feff of high spin type (5.1-5.8 Bohr magneton). In the Soret region the m.c.d, for the free haemin resembles fairly well that for the haemin-PLL at pH

POLYMER, 1974, Vol 15, June

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C.d. and m.c.d, of the haemin-poly(L-lysine) complex: $. Yamamoto et al. 8"7. This fact suggests that in spite of the low value of the magnetic susceptibility, the green complex has a very similar electronic state to that for the haemin without P L L (the high spin type). This means that the apparent low value of the magnetic m o m e n t for the green complex does not indicate a monomeric unit of a low spin haemin which show the very intense m.c.d. in the Soret region. Instead, it suggests that there exists some magnetic haeme-haeme interactions which make the tZeffvalue as low as that for a low spin type haemin.

CONCLUSION The direct relationship between the induced c.d. band magnitudes and the s-helix contents was shown for the h a e m i n - P L L complex in an aqueous solution. The m.c.d, spectra for the h a e m i n - P L L complex were

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explained from the established nature of the haemin and its complexes, and were used to elucidate the structure and electronic state of the h a e m i n - P L L complex. REFERENCES 1 Blauer, G. Nature 1961, 189, 396 2 Blauer, G. and Ehrenberg, A. Acta Chem. Scand. 1963, 17, 8 3 Blauer, G. Biochim. Biophys. Acta 1964, 79, 547 4 Blauer, G. and Ehrenberg, A. ibid. 1966, 122, 496 5 Blauer, G. and Zvilichousky, B. ibid. 1970, 221,442 6 Stryer, L. ibid. 1261, 54, 397 7 Beychok, S. 'Poly-a-amino acids', (Ed. G. D. Fasman), Marcel Dekker, New York, 1967, p 499 8 (a) Buckingham, A. D. and Stephens, P. J. A. Rev. Phys. Chem. 1966, 17, 399 (b) Schatz, P. N. and McCaffery, A. J. Q. Rev. Chem. Soc. 1969, 23, 552 9 Hatano, M. and Yoneyama, M. J. Am. Chem. Soc. 1970, 92, 1392