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J . Phys. Chem. 1987, 91, 176-179
Photdnduced Electron Transfer from Amino Acids and Protefns to 4-Nitroqulnoiine 1-Oxide in Aqueous Solutions Hiroshi Seki,* Akiko Takematsu, and Shigeyoshi Arai The Institute of Physical and Chemical Research, Wako-shi, Saitama 351 -01, Japan (Received: January 16, 1986; In Final Form: May 30, 1986)
The reactions of the triplet state of 4-nitroquinoline 1-oxide (4NQO) with a series of amino acids and some proteins in aqueous solutions have been studied by using a laser flash technique. Only tryptophan (TrpH) and tyrosine (TyrOH) among a series of amino acids quench the triplet 4NQ0 (T4NQ0) at a diffusion-controlled rate. Lysozyme, ribonuclease, and histone, which contain TrpH and/or TyrOH residues, had rate constants comparable to those of TrpH and TyrOH. The formation of the H adduct of 4 N Q 0 (4NQOH.1, which may be produced by the reaction of 4NQO- with water, was confirmed from the transient absorption spectra for 4NQ0 solutions containing these quenchers. The transient absorption spectra observed for TrpH and TyrOH solutions elucidated the formation of the deprotonated forms of TrpH' and TyrOH' (Trp' and Tyro') together with 4NQOH'. The result demonstrates that the electron transfer from TrpH or TyrOH to T4NQ0 occurs in the triplet quenching by TrpH or TyrOH. Since the almost same transient spectra as Trp' and Tyro' were observed for lysozyme and ribonuclease solutions, respectively, TrpH residues on lysozyme and TyrOH residues on ribonuclease are main quenching sites, where electron transfer and deprotonation occur. The quantum yields of T4NQ0,4NQOH', Trp', and T y r o produced by the excitation of the 4 N Q 0 solution containing TrpH or TyrOH with a 355-nm light pulse were determined to be 0.46, 0.47,0.41, and 0.41, respectively. The result shows that the efficiency in electron transfer from TrpH or TyrOH to T4NQ0 is -90%. For the reaction of T4NQ0 with methionine, arginine, histidine, lysozyme, or ribonuclease, the efficiency in electron transfer was also estimated to be nearly equal to that for the reaction with TrpH or TyrOH.
Introduction
4-Nitroquinoline 1-oxide (4NQO), one of the typical carcinogenic compounds, has the property to bind DNA bases via charge-transfer interaction in an aqueous The property may be closely related to carcinogenicity. The genetic function which DNA possesses emerges by forming chromatin, a complex of DNA and proteins such as histone and nonhistone chromosomal proteins. Such proteins bonded to DNA, as well as DNA and nucleic acid bases, also may participate in the process of carcinogenicity induced by chemicals. It, therefore, is worthwhile in the study of the reaction of 4 N Q 0 with chromatin to elucidate the reactions of 4NQO with amino acids and proteins. We have previously studied the photochemical reactions of 4 N Q 0 with D N A bases and related compounds as well as DNA in aqueous solutions by using a laser flash photolysis t e c h n i q ~ e . ~The triplet state of 4 N Q 0 (T4NQ0) produced by excitation with a 355-nm light pulse has been found to be efficiently quenched by the compounds having lower oxidation potentials. The transient absorption spectra have confirmed the formation of the H adduct radical of 4 N Q 0 (4NQOH') in photoirradiated solutions of 4 N Q 0 and the quenchers. These results suggested the participation of charge transfer in the reaction of T 4 N Q 0 with the quenchers, because 4NQOH' is considered to be produced by the reaction of 4NQO- with H20.3 However, evidence for the formation of any quencher cation was not obtained. One of the primary purposes of the present work is to find definitive proof of whether electron transfer occurs in the quenching of T 4 N Q 0 and to clarify the quenching mechanism by the determination of the quantum yields of the transients produced in the irradiated solutions. Our attention in the present work is especially focused on tryptophan (TrpH), tyrosine (TyrOH), and the proteins including these amino acids, because TrpH and TyrOH are easily oxidized to their cations or deprotonated forms of the cations, which have the characteristic absorption spectra, by UV-light (1) Nagata, C.; Kodama, M.; Tagashira, Y.; Immamura, A. Biopolymers 1966, 4,409-427. (2) Okano, T.; Uekama, K. Chem. Pharm. Bull. 1967, IS, 1812-1815. (3) Kasama, K.; Takematsu, A.; Yamamoto, S.; Arai, S . J . Phys. Chem. 1984,88, 4918-4921. (4) Dudley, Bryant, F.; Santus, R.; Grossweiner, L. I. J . Phys. Chem. 1975, 79. 2711-2716.
i r r a d i a t i ~ n ~or- ~the reactions with Br2*-. (SCN)2'-, SO4'-, and N3'.'-I0 Experimental Section
4 N Q 0 was purchased from Sigma Chemical Co. and recrystallized from acetone solutions. Analytical reagent grade amino acids from Wako Pure Chemical Industry Ltd. and Tokyo Kasei Kogyo Co. were used as received. Ribonuclease A (from bovine pancreas), protamine phosphate (from salmon sperm) and histone H2A (from calf thymus) were supplied by Sigma Chemical Co. Lysozyme (from egg white), crystallized 6 times, was obtained from Seikagaku Kogyo Co. They were used without purification. Bityrosine, used as the standard, was prepared by an enzymatic method and was purified by the Lehrer-Fasman procedure.'' Solutions were prepared by using redistilled water and buffered at pH 8 by employing phosphates, except for histone solutions, where sodium hydroxide was used. The concentrations are 1.O-1.4 X mol dm-3 for 4 N Q 0 and 5 X 10-5-1 X lo-* mol dm-3 for amino acids or proteins. Deaeration was carried out by a freezethaw cycle method for amino acid solutions and by blowing argon gas over the surface of the liquid in the cells for protein solutions. A conventional laser flash photolysis technique was used for the measurements of the absorption spectra and the decay rates of the transients. Excitation light was the third harmonic (355 nm) from a Nd:YAG laser (204s pulse duration). The detection system consisted of a 150-W Ushio xenon lamp as an analyzing light source, a monochromator (Ritsu Model MC-20N), a photomultiplier (Hamamatsu R758), and an oscilloscope (Tektronix Model 7904). The optical path lengths of a reaction cell were 10 mm. The details of the procedures for detecting transient absorption have been described previ~usly.~The apparatus and procedures used in pulse radiolysis have been also described ( 5 ) Feitelson, J.; Hayon, E. J . Phys. Chem. 1973, 77, 10-15. (6) Baugher, J. F.;Grosswdner, L. I. J. Phys. Chem. 1977, 81, 1349-1354. (7) Redpath, J. L.; Santus, R.; Ovadia, J.; Grossweiner,L. I. Int. J . Radiat. Biol. 1975, 27, 201-204.
(8) Posener, M. L.; Adams, G. E.; Wardman, P.; Cundall, R. B. J. Chem. Soc., Faraday Trans. 1 1976, 72, 2231-2239. (9) Land, E. J.; PrUtz, W. A. Int. J. Radiat. Biol. 1977, 32, 203-207. (10) Bansal, K. M.; Fessenden, R. W. Radiat. Res. 1976, 67, 1-8. (11) Lehrer, S. S.; Fasman, G. D. Biochemistry 1967, 6, 757-761. (12) Kira, A.; Arai, S.; Imamura, M. J. Chem. Phys 1971,54,4890-4895.
0022-3654/87/2091-0176$01.50/0 0 1987 American Chemical Society
The Journal of Physical Chemistry, Vol. 91, No. 1, 1987 177
Photoinduced Electron Transfer from Amino Acids TABLE I: Quenching Rate Constants of T4NQ0 and Oxidation Potentials of Ouenchers quencher tryptophan tyrosine methionine arginine histidine proline phenylalanine glutamic acid leucine lysine glycine histone lysozyme ribonuclease protamine
k2/dm3 mol-' s-I (pH 8) 3.7 x 109 3.1 x 109 2.9 x 107 1.7 x 107 1.6 x 107 1.3 x 107 1 x 107 1 x 107 1 x 107
