Induction of circular dichroism of symmetrical porphyrins bound to random coil polypeptides in aqueous solutions

Induction of circular dichroism of symmetrical porphyrins bound to random coil polypeptides in aqueous solutions Takashi Nezu and Shoichi Ikeda Department of Chemistry, Faculty of Science, Nagoya University, Chikusa, Nagoya 464, Japan (Received 7 July 1992; revised 12 October 1992) Absorption spectra and c.d. spectra have been measured in the region of Soret transition for aqueous solutions of porphine-meso-tetra( 4-N-methylpyridinium) ( TMpyP) tosylate in the presence of poly-L-glutamic acid, and of sodium porphine-meso-tetra(4-benzenesulphonate) (TPPS) or sodium porphine-meso-tetra(4benzoate) (TPPC) in the presence of poly-L-lysine, all at high [ P ] / [ D ] ratios at neutral pH. The TMpyP poly-L-glutamic acid system shows essentially no hypochromism and negligibly weak induced c.d., while the TPPS-poly-L-lysine or TPPC-poly-L-lysine system exhibits stron9 hypochromism and stron 9 induced c.d. The former absorption band slightly shifts to red, but the latter shows a large blue shift. The primary interaction of the porphyrin with the polypeptide is their electrostatic binding. The difference in their interaction must arise from different degrees of hydrophobic interaction, and stronger interaction of TPPS or TPPC with poly-L-lysine would cause most of TPPS ions or all of TPPC ions bound on poly-L-lysine to dimerize, fix rigidly and couple together electronically on fully charged poly-L-lysine. Keywords: Induced circular dichroism; Soret transition; porphyrin; polypeptide; random coil

Introduction Porphyrin is an important prosthetic group of haem proteins such as haemoglobin and cytochromes. While it is usually present with a metal ion incorporated at the molecular centre, its direct interaction with protein must also play a significant role in the physiological environment. With these haem proteins, strong c.d. is induced at the Sorer transition, indicating strong electronic coupling between porphine and protein moieties. Recently we have reported that the Soret transition of symmetrical porphyrin ions becomes optically active when e-helical polypeptide having side chain oppositely charged to porphyrin is added to a large excess 1. That is, porphine-meso-tetra(4-N-methylpyridinium) (TMpyP) 2 salt shows a positive c.d. band at 415 nm and a stronger negative c.d. band at 442 nm, if helical poly-L-glutamic acid is present, and porphine-meso-tetra(4-benzenesulphonate) (TPPS) 3 salt shows a positive c.d. band at 416 nm and a negative c.d. band at 426 nm, if helical poly-L-lysine is present. The magnitude of molar ellipticities of the latter is more than three times as large as that of the former. The hypochromicity of the Soret band for the latter is also much stronger than for the former. Similar behaviour of absorption and c.d. spectra was observed for aqueous solutions of porphine-mesotetra(4-benzoate) ( T P P C ) 4 salt in the presence of poly-L-lysine. It was imagined that detailed mode of interaction of porphyrin with helical polypeptide would differ between the T M p y P poly-L-glutamic acid system and the T P P S - or T P P C poly-L-lysine system. In the present work we communicate a more distinct difference in the mode of interaction of porphyrin with 0141-8130/93/020101-03 © 1993 Butterworth-HeinemannLimited

polypeptide, as revealed in the induction of c.d. on the Soret transition, when the polypeptide is randomly coiled.

Experimental Porphine-meso-tetra ( 4-N-methylpyridinium ) tosylate, porphine-meso-tetra (4-benzenesulphonic acid) dihydrochloride and porphine-meso-tetra(4-benzoic acid) were purchased from Porphyrin Products, Inc., Logan, UT. Sodium poly-.L-glutamate and poly-L-lysine hydrochloride were obtained from Protein Research Foundation, Minoh, Osaka. Both samples of the polypeptides had the average molecular weight higher than 13000, as measured by light scattering. The mixing molar ratio of amino acid residue to porphyrin, [ P ] / [ D ] , was 100 in most cases, with the porphyrin concentration, [D1, kept at 1.0 × 10-5 ~. The pH of solutions was adjusted by adding 0.1 N HC1 or 0.1 N NaOH. Absorption spectra were recorded on a Shimadzu UV-2200 spectrophotometer, using a 0.5 cm quartz cell, and c.d. spectra were measured on a Jasco J-40 A circular dichrometer, using a 0.5 cm quartz cell. Temperature was kept at 25°C. The pH was examined by a Horiba N-8F ion meter.

Results T M p y P has the Soret band at 421 nm with the molar absorption coefficient, ~ = 233 000, in aqueous solutions of pH from 3 to 10, while T P P S and T P P C have the Soret band at 413 and 414nm with e = 489000 and

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Porphyrin-polypeptide interaction: T. Nezu and S. Ikeda 10

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Figure 2 shows absorption spectra and c.d. spectra of aqueous solution of TPPC in the presence of poly-L-lysine. The absorption band shifts to blue, i.e. from 414 to 405nm, and is subject to strong hypochromism. These observations would suggest that TPPC ions bind to poly-L-lysine and form dimers or their aggregates on the random coil, in a similar way to TPPS. C.d. is also induced in the Soret region, and it is mainly characterized by a nearly conservative pair of positive band at 402 nm and a negative band at 417 nm. Bound TPPC ions would be all in the dimeric form and rigidly held on chiral random coils. The random coil form of poly-L-lysine is assured by the far ultraviolet c.d. spectra.

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486 000, respectively, in aqueous solutions of pH from 6.5 to 11. In the presence of poly-L-glutamic acid, the Soret band of aqueous solution of TMpyP locates at 425 nm but its molar absorption coefficient remains nearly unaltered. The weak red-shift indicates electrostatic binding of TMpyP on fully charged poly-L-glutamic acid, possibly assisted by weak hydrophobic effect. (More detailed examination of the spectral change with [P]/[D] shows that the Sorer band once weakens and then shifts to red with recovery of intensity at higher [P]/[D] ratioS.) However, only negligibly weak c.d. is induced on the Soret transition. This suggests that bound TMpyP ions are not rigidly fixed on the random coil. The random coil conformation of poly-L-glutamic acid is clear from the far ultraviolet c.d. spectra having a weak positive band at 216 nm and a strong negative band at 200 nm. Figure 1 shows absorption spectra and c.d. spectra of aqueous solution of TPPS in the presence of poly-Llysine. The hypochromism is large in the Sorer band and strong c.d. is induced on the Soret transition. Furthermore, the main absorption band shifts to blue, i.e. from 413 to 399 nm, and a weak shoulder appears at 415nm. This red-shifted band at 415nm becomes gradually stronger with increasing [P]/[D], and it can be attributed to a TPPS ion simply electrostatically bound to poly-L-lysine 6. On the other hand, the blue-shifted main band can be attributed to dimeric TPPS ions bound to poly-L-lysine, as will be referred to below. The induced c.d. is complicated, and both dimeric and monomeric bands are associated with many c.d. bands. This means that the dimeric and monomeric TPPS ions are rigidly bound to a random coil and electronically coupled together. Far ultraviolet c.d. spectra indicate that poly-L-lysine is randomly coiled even in the presence of TPPS.

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Table 1 compares the spectral data of the three porphyrin-random coil polypeptide systems. Both TMpyP and TPPS are tetravalent strong electrolytes. Carboxyl groups of TPPC are also nearly fully ionized at neutral pH, since the absorption spectra of free TPPC change sharply below pH 6 but remain unaltered over the region of pH from 6.5 to 11. The structural difference between the three systems lies in the chemical nature of ionic groups of porphyrin and side chains of polypeptide. Poly-L-lysine has a longer side chain and is more hydrophobic than poly-L-glutamic acid 7. Together with this, and owing to no tendency for dimerization of TMpyP in water, we may infer that hydrophobic interaction is stronger in the TPPS-poly-L-lysine or in the TPPC-poly-L-lysine system than in the TMpyP-polyL-glutamic acid system. TPPS ions have some tendency to dimerize in water, suggesting their higher hydrophobicitya-l°. Actually, the Soret band of free TPPS gradually weakens with increasing TPPS concentration, and in the presence of 7... O

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Porphyrin polypeptide interaction: T. Nezu and S. Ikeda Table 1 Spectral parameters for water-soluble porphyrin random coil polypeptide systems ([D] = 1.0 × 10 5 M) System

(nm)

eo (1 mo1-1 cm 1)

2 (nm)

[0o] (degcm 2 dmol 1)

TMpyP-poly-L-glutamic acid [ P ] / [ D ] 100 pH 7.33

405 425

87 500 232000

TPPS-poly-L-lysine [PJ/[D] 100 pH 7.47

399 415

159 000 80000

392 402 417 425 434 445

90 000 - 212 000 - 8 000a - 22 000a - 118 000 - 83 000

TPPC-poly-L-lysine [PI/[D] 100 pH 7.61

405

119000

402 417 448

63 000 -64000 12 000

a The band appears convex upward

NaC1 at higher concentration, it undergoes a larger blue-shift, locating at 408 nm in 1 M NaC1. Then T P P S ions are mostly in the dimeric form or its higher aggregates on random coil poly-L-lysine. Similarly, the Soret band of free T P P C is blue-shifted to 410 nm in the presence of 1 M NaC1. T P P C would also be in the dimeric form on random coil poly-L-lysine. Possibly because of the lower hydrophilicity of the carboxylate group than the sulphonate group, TPPC would have a somewhat higher tendency for dimerization or aggregation, compared with TPPS. As a result, T P P C ions are all in the dimeric form, giving the Soret band at 405 nm alone, while T P P S ions are mostly dimeric but partially monomeric, giving the bands at 399 and 415 nm, when they are bound to fully charged poly-L-lysine. T M p y P ions would be bound to a poly-L-glutamic acid chain sparsely, so that the effect of binding on the Soret transition is apparently weak, simply causing the absorption band to shift to red. The Soret transition remains essentially optically inactive, owing to the weak disymmetric effect. The largely blue-shifted Soret band of T P P S indicates that most of T P P S ions bound to poly-L-lysine are subject to strong electronic perturbation from other bound T P P S ions, which eventually leads to formation of dimeric T P P S ions or their array on fully charged poly-L-lysine. Dimerization of dye molecules upon binding to polypeptide or polymer was observed for the acridine orange-poly-L-glutamic acid system 11, for the acridine orange-poly-S-carboxymethyl-L-cysteine system 12, for the methyl orange-poly-L-lysine system 13,14, and for the crystal violet-polymethacrylic acid system ~5'16, all of which caused the absorption band of dye to shift largely to blue. The electronic coupling of bound dimers of T P P S with its bound monomers or dimers could assist further blue-shift of the Soret band. The induced c.d. of T P P S in the presence of random coil poly-L-lysine consists of complicated bands overlapped together, so that it may be attributed to

disymmetric electronic coupling of bound T P P S ions, both dimeric and monomeric, on a chiral random coil. The assignment of the blue-shifted and the red-shifted bands of T P P S in the presence of poly-L-lysine is in conformity with the dependence of the molar absorption coefficient of the red-shifted band at 415 nm on the [P]/[D] ratio 6. Similar consideration would hold for the T P P C - p o l y L-lysine system. Owing to the absence of the monomeric form of T P P C bound to poly-L-lysine, however, the TPPC-poly-L-lysine system exhibits a simpler feature of induced c.d., compared with the TPPS-poly-L-lysine system. Nevertheless, the magnitude of induced c.d. is stronger for the TPPS-poly-L-lysine system than for the TPPC-poly-L-lysine system.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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