Interaction of 4-Nitroquinoline-1-oxide with Indole Derivatives and

Chemical Sciences DiVision, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata 700 064, India. ReceiVed: October 19, 2005; In Final Form: M...
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J. Phys. Chem. B 2006, 110, 8850-8855

Interaction of 4-Nitroquinoline-1-oxide with Indole Derivatives and Some Related Biomolecules: A Study with Magnetic Field Sharmistha Dutta Choudhury and Samita Basu* Chemical Sciences DiVision, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata 700 064, India ReceiVed: October 19, 2005; In Final Form: March 9, 2006

Laser flash photolysis and an external magnetic field have been used for the study of the interaction of 4-nitroquinoline-1-oxide (4NQO) with some indole derivatives, amino acids, tyrosine and tryptophan, and model proteins, lysozyme and bovine serum albumin. In an aprotic medium, photoinduced electron transfer (PET) from indoles to 4NQO is accompanied by proton transfer from the indole moieties irrespective of the substitution at the N-1 position. For 1,2-dimethylindole, however, proton abstraction is hindered possibly due to steric effects. In a protic medium, obviously proton transfer is possible from the medium and is the dominating reaction following PET. The effect of an external magnetic field is very small for all the systems studied. This is attributed to a competition between geminate proton abstraction by the 4NQO radical anion from the partner radical cation and escape of the 4NQO radical anion to the medium followed by proton transfer. The latter process is more predominant, and the former one, which produces a small population of geminate spin-correlated radical pairs, leads to a minor field effect. Another interesting observation is the affinity of 4NQO toward the tryptophan residues in a protein environment. It is seen that PET takes place preferably from the tryptophan residues rather than from the tyrosine residues.

Introduction 4-Nitroquinoline-1-oxide (4NQO) is a model carcinogen that causes DNA damage like ionizing radiation and UV light.1,2 The carcinogenicity of 4NQO was first discovered by Nakahara et al. in skin painting experiments with mice.1 4NQO has also been shown to stack with the bases in DNA and form charge transfer complexes, and this property is thought to be closely related to its carcinogenicity.3 Molecular mechanical studies have also been performed for complexes between 4NQO and dinucleoside phosphates.4 Arai and co-workers have reported photoinduced electron transfer (PET) from DNA and related compounds to 4NQO.5 Since proteins bonded to DNA may also participate in the process of carcinogenicity induced by chemicals, the PET reactions of 4NQO with amino acids and proteins have also been elucidated.6 However, the radical ion species formed due to PET could not be directly observed in these studies due to subsequent protonation reactions. In this paper we report the direct observation of PET from several indole derivatives, which may be considered as a model for the tryptophan molecule, to 4NQO in acetonitrile (ACN) medium. Interesting differences have been observed in the behavior of the indole derivatives depending on their substitution. In ACN, PET is also accompanied by subsequent proton transfer from indoles. However, in protic medium protonation from the surrounding medium is the dominating reaction following PET. Now PET reactions lead to the formation of radical ion pairs (RIPs)/radical pairs (RPs), and in general can be affected by an external magnetic field (MF).7-9 The MF effect arises due to competition between electron spin dynamics and radical separation. The application of an external MF results in Zeemann splitting of the triplet sublevels, which in turn slows down the * Author to whom correspondence should be addressed. Phone: +91 033 23375345. Fax: +91 033 23374637. E-mail: [email protected].

intersystem crossing process thereby increasing the population of the initial spin state. It should be mentioned here that for triplet-derived RIPs/RPs the use of a heterogeneous medium is necessary to observe MF effects to prolong the lifetime of the RIPs/RPs such that they can retain their geminate character for a sufficiently long time for spin flipping to occur.7 So, for a triplet-generated RP, an external MF can increase the triplet population and hence increase radical escape. Thus the MF can perturb the free radical concentration in a system. This has immense significance in free-radical-based biochemical reactions. Indirectly, the MF can act as a co-carcinogen by enhancing the genotoxic potential of the radical carcinogen.10 In this context, we have also studied the effect of an external MF on the interaction between 4NQO and the indoles. The MF effect also serves as a tool for elucidating the mechanism of the interaction. We have also extended our investigation to the amino acids tyrosine (TyrH) and tryptophan (TrpH) and the proteins lysozyme and bovine serum albumin (BSA) to gain some insight into the nature of the interaction. Experimental Section Materials. 4NQO was obtained from Sigma. Indole (InH) was obtained from SRL, India, and N-methylindole (NMInH), 2-methylindole (2MInH), and 1,2-dimethylindole (DMInH) were obtained from Aldrich. UV spectroscopy grade solvent, ACN, was obtained from Spectrochem and was used as such without further distillation. Sodium dodecyl sulfate (SDS), TyrH, and TrpH were obtained from Sigma. Tris buffer, lysozyme, and BSA were obtained from SRL. Water was triply distilled. Methods. Transient absorption spectra were measured using a nanosecond flash photolysis setup (Applied Photophysics) containing an Nd:YAG laser (DCR-II, Spectra Physics). The

10.1021/jp055971l CCC: $33.50 © 2006 American Chemical Society Published on Web 04/07/2006

Interaction of 4NQO with Indole Derivatives

J. Phys. Chem. B, Vol. 110, No. 17, 2006 8851 TABLE 1: Quenching Rate Constants of 34NQO by Indole Derivatives in ACN and by TyrH, TrpH, Lysozyme, and BSA in Tris Buffer at pH 7.4 quencher

kq (M-1 s-1)

InH NMInH 2MInH DMInH TyrH TrpH lysozyme BSA

1.1 ((0.2) × 108 1.4 ((0.2) × 108 3.1 ((0.5) × 108 2.2 ((0.5) × 108 3.2 ((0.2) × 109 3.5 ((0.2) × 109 1.7 ((0.2) × 109 5.6 ((0.2) × 108

from the indole moieties to 4NQO•- ensues PET. So the reaction schemes can be represented as 3

Figure 1. Transient absorption spectra of 4NQO (1 × 10-4 M) (×), 4NQO (1 × 10-4 M)-InH (4 × 10-2 M) (b), 4NQO (1 × 10-4 M)NMInH (4 × 10-2 M) (2), 4NQO (1 × 10-4 M)-2MInH (4 × 10-2 M) (1), and 4NQO (1 × 10-4 M)-DMInH (4 × 10-2 M) (9) in ACN at 0.8 µs after the laser flash with excitation wavelength 355 nm. The inset shows the transient absorption spectra of 4NQO (1 × 10-4 M) (×) and 4NQO (1 × 10-4 M)-InH (4 × 10-2M) (b) at 2 µs after the laser flash.

sample was excited by 355 nm laser light (full width at halfmaximum of 8 ns). Transients were monitored through absorption of light from a pulsed Xe lamp (250 W). The photomultiplier (IP28) output was fed into a Tektronix oscilloscope (TDS 3054B, 500 MHz, 5 GS/s), and the data was transferred to a computer using TekVISA software. The MF effect (∼0.1 T) on the transient spectra was studied by passing direct current through a pair of electromagnetic coils placed inside the sample chamber. The samples were deaerated by passing pure Ar gas for 20 min prior to experiment. No degradation of the samples was observed during the experiment. Results and Discussion Figure 1 shows the transient absorption spectra of pure 4NQO (1 × 10-4 M) and 4NQO (1 × 10-4 M) in the presence of the indoles (4 × 10-2 M) InH, NMInH, 2MInH, and DMInH, in ACN medium at 0.8 µs after the laser flash at 355 nm. The maxima around 420 and 560 nm for pure 4NQO correspond to the triplet-triplet absorption of 4NQO.5,6,11,12 On addition of the indoles, the natures of the transient absorption spectra change, and new peaks are observed around 440 and 500 nm for all of the derivatives. However, the 500 nm peak is more prominent compared to the small shoulder around 440 nm for DMInH. It is known from literature that the radical anion of 4NQO, 4NQO•-, absorbs around 500 nm.11,12 So the appearance of the 500 nm peak in the presence of the indoles confirms the occurrence of PET from the indoles to 4NQO. This PET should also lead to the formation of indole radical cations, which absorb around 580 nm,13 and these are observed for all of the indole derivatives at a greater time delay (2 µs). This is because at a shorter time delay the absorption of indole radical cations is masked by 4NQO absorption. The inset to Figure 1 shows the absorption for 4NQO (1 × 10-4 M)-InH (4 × 10-2 M) at 2 µs, and here the absorption by In•+ at 580 nm is clearly observed. The 440 nm peak corresponds to the radical, 4NQOH•, which is formed by protonation of 4NQO•-.5,6 Thus proton transfer

4NQO* + XH f 4NQO•- + XH•+

(1)

(XH represents the indoles.) 4NQO•- + XH•+ f 4NQOH• + X•

(2)

Now InH and 2MInH have a labile N-H group so that proton abstraction from these derivatives can be very facile, leading to the absorption at 440 nm. The corresponding absorption by the indole radical ions should be observed around 520 nm but are masked by the radical cation absorptions in our case. Earlier Encinas et al. had also observed that for indoles unsubstituted at the N-1 position PET from indoles to anthracene was followed by proton transfer.14 But the appearance of the absorption at 440 nm for NMInH and DMInH in our case was very surprising. McGimpsey et al. have, however, reported that the chemistry of the cation radical of InH and NMInH are very similar, especially with respect to the deprotonation of the cation radical, and they suggested deprotonation at N-methyl to give a relatively stable indolylmethyl radical.15 This explains the observation of the 440 nm peak for NMInH. According to this explanation, a similar result also should have been obtained for DMInH, but in this case PET is the predominant reaction, and the subsequent proton abstraction from DMInH is a minor event leading to a small hump around 440 nm. This may be due to steric effects. Because of the presence of two methyl groups in DMInH, close approach toward 4NQO may be difficult, and a favorable orientation for abstraction of a proton probably cannot be attained. Linear fits were obtained for the plots of decay rates of triplet 4NQO against concentrations of the indoles, and the slopes correspond to the rate constant for the reaction of triplet 4NQO with the indole derivatives. Similar values were obtained for all of the derivatives, suggesting that PET is the main mode of interaction in all cases. The quenching constants are presented in Table 1. Figure 2 shows the transient absorption spectra for pure 4NQO (1 × 10-4 M) and 4NQO (1 × 10-4 M) in the presence of indoles (4 × 10-2 M) in 10% SDS micellar medium. The spectrum for pure 4NQO is slightly shifted compared to that in ACN, and the peaks appear around 410 and 580 nm. This is characteristic of the polar N-O bond in quinoline-N-oxides.5 On addition of the indoles, in this case, it is observed that the 500 nm peak corresponding to 4NQO•- is completely absent, and only the 440 nm peak due to 4NQOH• is observed. This is expected because now in addition to proton abstraction from the indoles proton transfer is also possible from the medium itself, and so all 4NQO•- are converted to 4NQOH•. The corresponding absorption by the indole radical ions can also be detected as a small hump around 520 nm.13

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Dutta Choudhury and Basu the RPs are initially formed in the triplet spin state (Figure 3). The MF effect is quite small but consistent and has been confirmed by repeated experiments. To demonstrate this consistency, the MF effects for all of the derivatives are shown in Figure 3 despite their similarity. The small magnitude of the MF effect can be explained by considering that only those geminate RPs that are formed by proton abstraction from the indole moieties are capable of responding to a MF. Randomly generated radicals formed by proton abstraction from the medium do not respond to an external MF. Thus 3

(4NQO•- XH•+) f 3(4NQOH• X•) T 1(4NQOH• X•)

(3)

(MF effect) (4NQO•- XH•+) f 4NQOH• + XH•+

3

Figure 2. Transient absorption spectra of 4NQO (1 × 10-4 M) (×), 4NQO (1 × 10-4 M)-InH (4 × 10-2 M) (b), 4NQO (1 × 10-4 M)NMInH (4 × 10-2 M) (2), 4NQO (1 × 10-4 M)-2MInH (4 × 10-2 M) (1), and 4NQO (1 × 10-4 M)-DMInH (4 × 10-2 M) (9) in 10% SDS at 0.8 µs after the laser flash with excitation wavelength 355 nm.

On application of an external MF, the absorption around 440 nm is enhanced for all of the indole derivatives, indicating that

(escape, no MF effect)

(4)

Figure 4 shows the transient absorption spectra of 4NQO (1 × 10-4 M) in the presence of TyrH and TrpH (both 3 × 10-3 M) in 10% SDS micelles. The spectra are in accordance with that observed earlier in aqueous phosphate buffer solution.6 Recently an ab initio study of the hydrogen bonding interaction and PET between 4NQO and TrpH has also been reported.16

Figure 3. Transient absorption spectra of (A) 4NQO (1 × 10-4 M)-InH (4 × 10-2 M) at 0 T (b) and 0.1 T (O), (B) 4NQO (1 × 10-4 M)NMInH (4 × 10-2 M) at 0 T (2) and 0.1 T (4), (C) 4NQO (1 × 10-4 M)-2MInH (4 × 10-2 M) at 0 T (1) and 0.1 T (3), and (D) 4NQO (1 × 10-4 M)-DMInH (4 × 10-2 M) at 0 T (9) and 0.1 T (0) in 10% SDS at 0.8 µs after the laser flash at 355 nm.

Interaction of 4NQO with Indole Derivatives

J. Phys. Chem. B, Vol. 110, No. 17, 2006 8853

Figure 4. Transient absorption spectra of 4NQO (1 × 10-4 M) (×), 4NQO (1 × 10-4 M)-TyrH (3 × 10-3 M) (9), and 4NQO (1 × 10-4 M)-TrpH (3 × 10-3 M) (b) in 10% SDS, 2 µs after the laser flash with excitation wavelength 355 nm.

The 440 nm absorption in Figure 4 corresponds to 4NQOH•, the hump around 520 nm for 4NQO-TrpH corresponds to the TrpH radical (Trp•),17 and the slight shoulder around 410 nm for 4NQO-TyrH corresponds to the TyrH radical (Tyr•).18 The amino acid radicals are formed by deprotonation of the radical cations. It should be mentioned here that in ACN medium only quenching of 4NQO was observed on addition of TyrH and TrpH, and no new peaks could be detected due to the poor solubility of the amino acids. In the presence of an external MF, there is a small enhancement around the 440 nm peak similar to that observed for the indoles. This MF effect arises due to the coupling of spin and diffusion dynamics of the spin-correlated RPs, 1,3(4NQOH• Tyr•) and 1,3(4NQOH• Trp•) (Figures 5a and 5b, respectively). A corresponding increase should have been obtained around 520 nm for the TrpH system and around 410 nm for the TyrH system but could not be detected in our case probably because of the weak absorptions in these regions. We have also studied the interaction of 4NQO with the model proteins lysozyme and BSA. The interaction of 4NQO with lysozyme has been studied earlier as an example of a protein that is closely related with nucleic acid in terms of physiological functions.6 BSA is also a very popular model protein for the study of interactions with small molecules and has been used to study PET reactions earlier.19-21 Moreover, at neutral pH lysozyme is positively charged (isoelectric point 11.4), and BSA is negatively charged (isoelectric point 4.2), and we were interested in seeing whether this charge difference has any influence on the interaction with 4NQO and the MF effect.22 Figure 6 shows the transient absorption spectra of 4NQO (1 × 10-4 M), 4NQO (1 × 10-4 M)-lysozyme (5 × 10-4 M), and 4NQO (1 × 10-4 M)-BSA (5 × 10-4 M) in Tris buffer at pH 7.4. The transient absorption for 4NQO-lysozyme corresponds with that observed earlier.6 It was seen that although the quenching rate constants of triplet 4NQO by TrpH and TyrH are similar only the TrpH residues participate in the PET reaction between lysozyme and 4NQO, and the transient absorption spectrum resembles that for the interaction with free TrpH. This was explained on the basis of steric hindrance for TyrH residues within lysozyme since the latter has 6 TrpH residues, some of which are on the protein surface, and 3 TyrH residues that are in the interior of the protein. In the present work it was observed

Figure 5. Transient absorption spectra of (A) 4NQO (1 × 10-4 M)TyrH (3 × 10-3 M) at 0 T (9) and 0.1 T (0) and (B) 4NQO (1 × 10-4 M)-TrpH (3 × 10-3 M) at 0 T (b) and 0.1 T (O) in 10% SDS, 1.6 µs after the laser flash with excitation wavelength 355 nm.

Figure 6. Transient absorption spectra of 4NQO (1 × 10-4 M) (×), 4NQO (1 × 10-4 M)-lysozyme (5 × 10-4 M) (b), and 4NQO (1 × 10-4 M)-BSA (5 × 10-4 M) (9) in Tris buffer at pH 7.4, 0.8 µs after the laser flash with excitation wavelength 355 nm.

that for BSA too the spectrum resembles that of free TrpH. This is surprising because albumins are characterized by a low content of TrpH.19 BSA has only 3 TrpH residues but 21 TyrH residues, and despite this PET takes place preferentially at the sites of

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Dutta Choudhury and Basu that has been attributed to Coulomb interaction.22 However, in our case, lysozyme and BSA responded similarly, and no differences were observed due to their opposite charges. This could be because 4NQO is initially a neutral molecule, and after PET the proton abstraction step is so fast that the effect of charge cannot be asserted. Mohtat et al. had also observed a small MF effect for the interaction of triplet benzophenone with BSA and human serum albumin.25 They explained this reduced effect to the poor confinement of the radicals by the protein environment compared to micelles where the confinement is efficient and the MF effect observed is also large. The poor confinement promotes radical separation, which is not sensitive to a MF. In our case, however, a consistently low MF effect was observed for all of the systems, and as explained earlier we feel that this is due to the small population of spin-correlated geminate RPs that are formed in the interaction, and only these are capable of responding to the MF. Conclusion It can be concluded that PET from indoles to 4NQO is accompanied by subsequent proton transfer irrespective of the substitution at the N-1 position. However steric effects can hinder the proton abstraction as shown for DMInH. A small MF effect is observed for the interaction of 4NQO with the indoles, TrpH, TyrH, lysozyme, and BSA. The small magnitude of the MF effect is attributed to the fact that a majority of the RPs are generated by random proton abstraction from the medium and only the few geminately generated spin-correlated RPs are sensitive to the external MF. It is also observed that in a protein environment the interaction of 4NQO occurs predominantly at the sites of the TrpH residues.

Figure 7. Transient absorption spectra of (A) 4NQO (1 × 10-4 M)lysozyme (5 × 10-4 M) at 0 T (b) and 0.1 T (O) and (B) 4NQO (1 × 10-4 M)-BSA (5 × 10-4 M) at 0 T (9) and 0.1 T (0) in Tris buffer at pH 7.4, 1.0 µs after the laser flash at 355 nm.

TrpH residues. Because of the lesser number of TrpH residues available, the transient absorption for 4NQO-BSA is less than that of 4NQO-lysozyme. This indicates that in a protein environment the 4NQO molecule has an affinity for the TrpH residues. Alternately, 4NQO is probably capable of destroying the native structure of the proteins such that the TrpH residues become more exposed and hence more susceptible to the attack. It was however observed that in a homogeneous mixture of 4NQO, TrpH, and TyrH the resultant transient absorption spectrum shows contribution from both the TrpH and the TyrH molecules. Table 1 also shows the quenching constants of triplet 4NQO by TyrH, TrpH, lysozyme, and BSA. The considerably lower value in the case of BSA shows that here the interaction takes place mainly with the TrpH residues, which are fewer in number. Now it has been mentioned earlier that to observe MF effects for triplet RPs it is necessary to confine the system in heterogeneous media such as micelles. Interaction between the proteins and 4NQO can also produce a confined RP, which makes the observation of MF effects possible. In the present study, a very small MF effect was observed for both 4NQOlysozyme and 4NQO-BSA around the 440 nm region (Figure 7). MF effects have been observed previously in protein environments.22-25 Recently a large MF effect has been observed for the flavin mononucleotide-hen egg white lysozyme system

Acknowledgment. We would like to thank Mrs. Chitra Raha for technical assistance and Ms. Asima Chakraborty for help. References and Notes (1) Nakahara, W.; Fukuoka, F.; Sugimura, T. Gann 1957, 48, 129. (2) Fann, Y. C.; Metosh-Dickey, C. A.; Winston, G. W.; Sygula, A.; Rao, D. N. R.; Kadiiska, M. B.; Mason, R. P. Chem. Res. Toxicol. 1999, 12, 450 and references therein. (3) Winkle, S. A.; Tinoco, I., Jr. Biochemistry 1979, 18, 3833. (4) Lybrand, T.; Dearing, A.; Weiner, P.; Kollman, P. Nucleic Acids Res. 1981, 9, 6995. (5) Kasama, K.; Takematsu, A.; Yamamoto, S.; Arai, S. J. Phys. Chem. 1984, 88, 4918. (6) Seki, H.; Takematsu, A.; Arai, S. J. Phys Chem. 1987, 91, 176. (7) Steiner U. E.; Ulrich, T. Chem. ReV. 1989, 89, 51 and references therein. (8) Bhattacharya K.; Chowdhury, M. Chem. ReV. 1993, 93, 507. (9) Gould I. R.; Turro, N. J.; Zimmt, M. B. AdV. Phys. Org. Chem. 1984, 20, 1. (10) Scaiano, J. C.; Cozens, F. L.; McLean, J. Photochem. Photobiol. 1994, 59, 585. (11) Shi, X.; Platz, M. S. J. Phys. Chem. A 2004, 108, 4385. (12) Ezumi, K.; Kubota, T.; Miyazaki, H.; Yamakawa, M. J. Phys. Chem. 1976, 80, 980. (13) Jovanovic, S. V.; Steenken, S. J. Phys. Chem. 1992, 96, 6674. (14) Encinas, M. V.; Previtali, C. M.; Bertolotti, S. J. Chem. Soc., Faraday Trans. 1996, 92, 17. (15) McGimpsey, W. G.; Go¨rner, H. Photochem. Photobiol. 1996, 64, 501. (16) Liu, J. F.; Li, X. Y.; Zhu, Q. Int. J. Quantum Chem. 2004, 98, 33. (17) Creed, D. Photochem. Photobiol. 1984, 39, 537. (18) Creed, D. Photochem. Photobiol. 1984, 39, 563. (19) Carter, D. C.; Ho, J. X. AdV. Protein Chem. 1994, 45, 153. (20) Boch. R.; Mohtat, N.; Lear, Y.; Arnason, J. T.; Durst, T.; Scaiano, J. C. Photochem. Photobiol. 1996, 64, 92.

Interaction of 4NQO with Indole Derivatives (21) Bhasikuttan, A. C.; Sapre, A. V.; Shastri, L. V. J. Photochem. Photobiol., A 2002, 150, 59. (22) Miura, T.; Maeda, K.; Arai, T. J. Phys. Chem. B 2003, 107, 6474. (23) Harkins, T. T.; Grissom, C. B. Science 1994, 263, 958.

J. Phys. Chem. B, Vol. 110, No. 17, 2006 8855 (24) Taraban, M. B.; Anderson, M. A.; Grissom C. B. J. Am. Chem. Soc. 1997, 119, 5768. (25) Mohtat, N.; Cozens, F. L.; Hancock-Chen, T.; Scaiano, J. C.; McLean; J.; Kim, J. Photochem. Photobiol. 1998, 67, 111.