J. Phys. Chem. 1995,99, 14078-14082
14078
Reactions and Rate Constants between Hydroxyl Radicals and the Dimer and Monomer of Spin Trap 2-Methyl-2-nitrosopropaneDetermined by the Pulse Radiolysis Method Mikinori Kuwabara,**tShunsuke Miyake? Takashi Jin2 and Sadashi Sawamura' Department of Radiation Biology, Faculty of Veterinary Medicine, Department of Nuclear Engineering, Faculty of Engineering, and Institute for Electronic Science, Hokkaido University, Sapporo 060, Japan Received: March IO, 1995; In Final Form: June 23, I995@
Reaction rate constants between OH radicals and the dimer and monomer of the spin trap 2-methyl-2nitrosopropane ( M N P ) were determined by the pulse radiolysis method. Before pulse radiolysis experiments, the molar absorbance coefficients of the dimer and monomer were determined by a method combining NMR and optical absorbance spectrometries. First, NMR signal intensities of the dimer and monomer were related to their molar concentrations using deuterated tert-butyl alcohol, (CH3)3C-OD, as a standard. Since the monomer was eliminated by bubbling the solution with Ar gas and the remaining dimer was found to gradually decompose to the monomer, the changes of molar concentrations against time starting from the dimer alone until reaching dimer-monomer equilibrium were next measured by monitoring both NMR and optical absorbance spectra (at 6 = 1.59 and A = 320 nm for the dimer and at 6 = 1.28 and 1 = 662 nm for the monomer). The molar absorbance coefficients were finally determined to be 6320 ",,, = 410 f 40 M-' cm-' for the dimer and 6662 ",,, = 10 f 5 M-' cm-I for the monomer by comparing the curves measured by NMR with those measured by optical absorbance spectrometry. Pulse radiolysis experiments on the reaction rate constant between OH radicals and the dimer were carried out in aqueous solutions containing different amounts of the MNP dimer alone, and it was determined to be (3.0 f 0.3) x lo9 M-' s-I. Experiments on the reaction rate constant between OH radicals and the monomer were carried out in solutions containing both the dimer and monomer, and it was determined to be (1.3 f 0.9) x 1O'O M-' s-'.
1. Introduction The spin trapping method is now widely applied in biology and medicine and utilized to trap and identify not only reactive oxygens such as OH radicals and superoxide (02-)' but also oxygen radical-induced radicals of cellular components such as DNA,2-5 protein^,^^^ and lipid^.^,^ Of the several spin traps, nitroso spin trap 2-methyl-2-nitrosopropane (MNP) is a compound suitable to exactly assign the structures of oxygen radicalinduced radicals. However, there are some problems with this compound, especially for its application to quantitative measurements of radicals. First, it takes two forms in aqueous solutions (dimer and monomer). MNP exists only in the dimer form in the solid state. When the soid is dissolved in an aqueous solution, the dimer gradually decomposes to the monomer to reach the dimer-monomer equilibrium approximately 10 h after dissolving. While only the monomer form can serve as a spintrapping agent, an aqueous solution consisting of the monomer alone cannot be prepared. Furthermore, the monomer is volatile and is easily lost from solution. Second, this compound traps free radicals produced by its decomposition to form byproducts that complicate the radical assignments.l0%' These phenomena make the quantitative measurements of free radicals difficult. Therefore, not only is a method to exactly measure the concentrations of the dimer and monomer required, but it is also necessary to know reaction rate constants with OH radicals for application of the spin-trapping method to quantitative measurements of free radicals. In our previous study we reported that hydrated electrons (eaq-) produced by the radiolysis of water preferentially reacted with the dimer with a relatively
* To whom correspondence should be addressed. t
* @
Department of Radiation Biology. Department of Nuclear Engineering. Institute for Electronic Science. Abstract published in Aduance ACS Abstracts, September 1, 1995.
0022-365419512099-14078$09.0010
high reaction rate constant.I2 The present study was carried out to determine the reaction rate constants between OH radicals and the dimer and monomer using the pulse radiolysis method. Before pulse radiolysis experiments, the molar absorbance coefficients of the dimer and monomer were determined to ascertain their exact concentrations at the time pulse radiolysis experiments were performed. Then pulse radiolysis experiments were done for the dimer and monomer. Since the solution containing only the dimer could be obtained by bubbling with Ar gas for several minutes, experiments to determine the reaction rate constants between OH radicals and the dimer were carried out immediately after bubbling with N20 gas. For the reaction rate constant between OH radicals and the monomer, since aqueous solutions containing the monomer alone could not be prepared, experiments were carried out on solutions containing both the dimer and monomer, which were obtained by standing the solution for adequate intervals after dissolving.
2. Experimental Section 2.1. Materials. The spin trap 2-methyl-2-nitrosopropane (MNP) was purchased from Sigma Chemical Co. (St. Louis, MO). Deuterated tert-butyl alcohol, (CH3)3C-OD (98%), was obtained from Aldrich Chemical Co. (Milwaukee, WI). D20 (99.8%) was from Bio-Rad Laboratories, Inc. (Hercules, CA). All experiments were carried out using triply distilled water. 2.2. NMR Spectrometry. 'H-NMR spectra of the dimer and monomer of MNP were obtained by using a Varian XL200 spectrometer. MNP powder (2.25 mg) was added to 1 mL of D20. Air in the solution was exchanged for Ar gas by bubbling the solution for 5 min. The powder was dissolved by stirring the solution for 12 h in the dark. The MNP solution was transferred to an NMR cylindrial Pyrex tube and again bubbled with Ar gas to remove the monomer, and the tube was sealed. NMR spectra were recorded at 90 min intervals for 24 0 1995 American Chemical Society
J, Phys. Chem., Vol. 99, No. 38, 1995 14079
Reactions between OH Radicals and Spin Trap MNP h at room temperature in the dark. Each spectrum corresponded to four acquisitions. The chemical shift of the NMR spectrum was measured using 4,4-dimethyl-4-silapentanesodium sulfonate. The NMR signal intensity of (CH&C-OD was used to estimate the molar concentrations of the dimer and monomer of MNP. Prior to NMR measurements, D20 solution containing 0.1 M (CH&C-OD was bubbled with Ar gas for 5 min in an NMR Pyrex tube. The integrated value of the NMR signal was compared with those of the dimer and monomer of MNP. Since 0.2% HzO was present in DzO, the NMR signal intensity from H20 in each sample was used to calibrate the NMR signal intensities. Finally, the time dependence starting from the dimer alone until reaching the dimer-monomer equilibrium was obtained by measuring the NMR signal intensities of the dimer and monomer at 6 = 1.59 and 1.28, re~pectively,'~ and plotted as the changes in the molar concentrations against time after Ar bubbling. 2.3. Optical Absorbance Spectrophotometry. Optical absorbance spectra of the dimer and monomer of MNP were observed with a Hitachi U-2000 spectrophotometer. MNP powder was added to H20 in a flask at a concentration of 2.25 mg/mL. Air in the solution was exchanged for Ar gas by bubbling the solution for 5 min. The powder was dissolved by stirring the solution for 12 h in the dark. The MNP solution was pipetted into several optical cuvettes (1 x 1 x 4 cm), and each cuvette was bubbled with Ar gas to remove the monomer and sealed. The decrease of the dimer concentration and the increase of the monomer concentration were monitored at 2. = 320 and 662 nm, re~pectively,'~ at 90 min intervals for 24 h at room temperature. Since the light for absorbance measurements might decompose both the dimer and monomer to unknown products, the MNP solution was continually renewed prior to each measurement. 2.4. Determination of the Optical Absorbance Coefficients of the Dimer and Monomer of MNP. The changes in the molar concentrations of the dimer and monomer of MNP against time after Ar bubbling obtained by NMR spectrometry were compared with those in their optical absorbances obtained by spectrophotometry. After the fit of both curves was made, molar absorbance coefficients of the dimer and monomer were determined. 2.5. Pulse Radiolysis Experiments. Pulse radiolysis experiments were carried out using the pulse radiolysis system constructed by the staff of the Department of Nuclear Engineering, Faculty of Engineering, Hokkaido Univer~ity.'~Electron beams with a 50 ns pulse width were obtained from a 45 MeV electron linear accelerator, and the dose per pulse determined by Fricke's dosimetry was 80 Gy. The light for analysis was obtained by flashing a xenon lamp (1 kW) in a series of pulses and filtered to cut off wavelengths below 390 nm. The light was incident to the solution at a right angle to the electron beam and monochromatized at 475 nm, corresponding to the absorbance wavelength of (SCN)$- by the monochromator (Shimazu Monochromate SPG-100). The impressed voltage of the photomultiplier was 700 V. Electric data from the photomultiplier were stored at a digital storage oscilloscope (Philips M3320A) and then transferred to a computer system (NEC PC9801VM). Pulse generation, data storage into the oscilloscope, kinetic analysis of the light absorbance due to transient species, and printing were all performed by the computer system. The computer program was made by the staff of the Department of Nuclear Engineering, Faculty of Engineering, Hokkaido University. OH radicals were generated by irradiating an N20-saturated aqueous solution by electron pulses. Water radiolysis products,
eaq-, were converted to OH radicals by the reactions with N20.
H,O
ionizing radiation
+
eaq- N,O
OK,eaq-, H'
+ H,O - OH', OH-, N,
(1)
(2)
The reaction rate constants between OH radicals and the dimer and monomer were determined by observing, the competitive reactions of OH radicals among the dimer and monomer and SCN-.
+ SCN- -OH- + SCN' OH' + dimer -products kSCN-
OH'
(3)
kdimer
OH'
+ monomer -products 'monomer
(4) (5)
SCN' reacts further with SCN- to form (SCN)2'-, which is a short-lived species but detectable by light absorbance at 475 nm. After electron pulse irradiation, the light absorbance at 475 nm rapidly increases, then reaches the maximum, and gradually decreases. If the yield of OH radicals, G(OH'), corresponds to the maximum, we can assume that G(0H') = G(SCN') = G((SCN)z*-) in the absence of MNP, and the following equation can be obtained by applying the theory of competitive reaction to eqs 3, 4,and 5, G(0H') G((SCN),'3
=1+
+
kdimer[dimerl kmon,mer[monomerl
kScN- [SCN-]
k s c ~ -SCN-] [
(6) where G((SCN)i-) is the yield of (SCN)2*- in the presence of MNP, and [SCN-I, [dimer], and [monomer] are the concentrations of SCN-, dimer, and monomer, respectively. G(0H') and G((SCN)2*-) correspond to the maximum light absorbance at 475 nm due to (SCN)2'- in the presence or absence of MNP, respectively. When only the dimer is present, eq 6 is changed to eq 7.
From the slope of the experimentally obtained straight line, the ratio O f kdimer/kSCN- is obtained. Since ~ S C N -= 1.1 x 10" M-' s-',I6 the value of kdimer can be calculated. When both the dimer and monomer are present, eq 6 is changed to the following equation:
Since kdimer is now a known constant and the ratio of [dimer]/ [SCN-] is also experimentally obtained, kmonomer is calculated from the slope of the straight line experimentally obtained. The reaction rate constant between OH radicals and the dimer was measured by fixing the concentration of SCN- at 1 mM and varying the concentrations of MNP. MNP powder was dissolved in Ar-saturated aqueous solutions containing 1 mM SCN- by stirring for 12 h in quartz cells (1 x 1 x 4 cm). The solutions were then bubbled with N20 gas for 5 min to remove the monomer. Immediately after the molar concentrations of the dimer were measured by optical absorbance at 320 nm, pulse radiolysis experiments were carried out. The reaction rate
Kuwabara et al.
14080 J. Phys. Chem., Vol. 99, No. 38, 1995
-
0 0
5
10
15
20
25
Time (h)
(a)
2.5
0.08
2.0
E 1.5
2
....... .......
A
.....6...
A66znm
3"
P 0.04
1 .o
0
5
IO
15
20
25
Time (h)
(b)
6 0
50
40
30
20
0
IO
wm)
Figure 1. 'H NMR spectrum obtained after dissolving 2.25 mg of MNP powder in 1 mL of D20 solution by stimng it for 12 h in the dark, then bubbling it with Ar gas for 5 min, and storing it for 24 h in the dark. Peak a is a signal from 0.2%H20 in D20, and peaks b and c are signals from the dimer and the monomer, respectively.
constant between OH radicals and the monomer was measured in the same way, but the solution used was prepared by being stored at room temperature for 12 h after bubbling with N20 and measuring the molar concentrations of the monomer and dimer at 662 and 320 nm, respectively. To diminish the effects of light-induced products of MNP on the competitive reactions, every experiment was performed using newly exchanged solution. Experiments were repeated five times.
3. Results 3.1. Time Dependence of the Equilibrium between the Dimer and Monomer Observed by NMR Spectrometry. MNP is present in two forms between the dimer and monomer in aqueous solution as follows.
Figure 2. (a) Time dependence in the changes of molar concentrations of the dimer (A)and monomer (e)observed by 'HNMR spectrometry. NMR measurements were started just after bubbling with Argas for 5 min and repeated 16 times at intervals of 90 min. (b) Time dependence in the changes of optical absorbances at 320 nm (dimer, m) and 662 nm (monomer, +) observed by optical absorbance spectrometry. The MNP solution was continually renewed prior to each measurement.
NMR spectrometry at 90 min intervals for 24 h at room temperature in the dark. Figure 1 shows the NMR spectrum at 24 h. The signals of dimer (b) and monomer (c) were observed at 6 = 1.59 and 1.28 ppm, respectively. The highest peak (a) at 6 = 4.8 ppm corresponded to the NMR signal due to 0.2% contamination by H20 in D20. To assign the NMR signal intensities of the dimer and monomer to their molar concentrations, they were compared to the NMR signal intensity of 0.1 M (CH3)3COD in D20. The NMR signal due to H20 in D20 solution was utilized to correct all spectra to each other. Figure 2a shows the changes in the molar concentrations of the dimer and monomer of MNP against time after Ar bubbling which were obtained by NMR spectrometry. A gradual decrease of the dimer and increase of the monomer were observed. The molar concentration of the dimer at 0 h was 6.1 mM. The molar concentration of the dimer calculated theoretically from 2.25 m g / d corresponded to 12.9 mM of the dimer. This meant
CH3 2 H,C--k--N=O
I
CH3
f H,C-L--N+--N CH3
YH3
1 I + CH30
-CH, I CH,
Solid MNP is present in a dimer form and slightly soluble in water but takes a long time to completely dissolve. While the dimer dissolves, it gradually decomposes to the monomer to achieve dimer-monomer equilibrium. Since the monomer is volatile, this can easily be removed from the water by bubbling with an adequate gas. In the present study we first completely dissolved 2.25 mg of MNP in 1 mL of D20 solution by stirring for 12 h in the dark. The solution was bubbled once with Ar gas for 5 min to remove the monomer and then examined by
that about half of the dimer disappeared during preparation of the solutions. 3.2. Time Dependence of the Equilibrium between the Dimer and Monomer Observed by Spectrophotometry and Determination of Their Molar Absorption Coefficients. The time dependence in the changes of optical absorbances at 320 n"(dimer) and 662 nm (monomer) are plotted in Figure 2b. The concentration of the dimer decreased with increasing time, whereas the concentration of the monomer increased with increasing time, showing a time dependence similar to that obtained by NMR spectrometry. The dimer possessed its maximum absorbance at 287 nm, but the absorbance of the solution (2.25 mg/mL) at this wavelength was too strong to
Reactions between OH Radicals and Spin Trap MNP
J. Phys. Chem., Vol. 99, No. 38, 1995 14081
TABLE 1: Molar Absorbance Coefficients of the Dimer and Monomer of MNP and Their Reaction Rate Constants with OH Radicals dimer monomer molar absorbance 410 f 40 at 320 nm 10 f 5 at 662 nm coefficient ( E ) (M-I cm-I) reaction rate (3.0 f 0.3) x lo9 (1.3 f 0.9) x 1Olo constant ( k ) (M-I s-l) 0
1
2
3
4
5
6
[dimerl/[KSCNl (A)
0 0
0.2
0.6 0.8 1 (monomer]/(KSCN] (8)
0.4
1.2
1.4
Figure 3. (A) Plots of G[OH]/G[(SCN)2*'-]vs [dimer]/[KSCN]. (B) Plots of (G[OH]/G[(SCN)2'-] - &he,[dimer]/&sc~[KSCN]) vs [mono-
mer]/[KSCN]. The experiments were repeated five times. The error bars represent standard deviations. obtain data concerning its time dependence. When these curves were superimposed on those shown in Figure 2a, the curves obtained from the dimer were found to match each other fairly well, whereas the curves from the monomer were slightly different from each other at the initial increase, though better matching was observed at the time interval between 15 and 24 h. This is due to the fact that the absorbance at 662 nm due to the monomer was quite low, and therefore, slight aberrations were unavoidable. The molar absorbance coefficient of the dimer (e:?:,,) was obtained by fitting the decay curve of NMR to that of optical absorbance in the whole time interval measured and was determined to be 410 f 40 M-' cm-', and that of the monomer was obtained by fitting the curve of NMR to that of optical absorbance at the interval between 15 and 24 h and was determined to be 10 f 5 M-' cm-I. 3.3. Measurements of Reaction Rate Constants by the Pulse Radiolysis Method. 3.3.1. Reaction Rate Constant between OH Radicals and the MNP Dimer. Prior to pulse radiolysis experiments, aqueous solutions containing 1 mM SCN- and adequate amounts of MNP were prepared in quartz cells. Immediately after the solutions were bubbled with N20 gas for 5 min, the molar concentrations of the dimer in the solutions were measured by optical absorbance at 320 nm, and subsequently, pulse radiolysis experiments to determine the reaction rate constant between OH radicals and the MNP dimer were carried out on the solutions. According to eq 7, the ratios G(OH')/G((SCN)2'-) experimentally obtained were plotted against the concentration ratios [dimer]/[SCN-] (Figure 3A). A straight line was drawn by applying the least-squaresmethod. Since k s C N - = 1.1 x 1Olo M-I s-', the reaction rate constant between OH radicals and the dimer (kjlmer) was calculated from the slope of the line and was (3.0 & 0.3) x lo9 M-' s-I. 3.3.2. Reaction Rate Constant between OH Radicals and the MNP Monomer. For pulse radiolysis experiments to determine the reaction rate constant between OH radicals and the monomer, adequate amounts of MNP powder were first dissolved in aqueous solutions containing 1 mM SCN- and
stored for 12 h after bubbling with N20 to obtain the dimermonomer equilibrium. Then the molar concentrations of the dimer and monomer were measured. Equation 8 was used to analyze the experimental results. Since the kdimer was now a known constant, the second term of the left side in this equation could be calculated when the concentration of the dimer was measured. The results obtained from pulse radiolysis experiments are shown in Figure 3B. By applying the least-squares method to the experimentally obtained data, the slope of the line was determined, and the reaction rate constant between OH radicals and the monomer (kmonomer) was calculated to be (1.3 f 0.9) x 10'" M-' s-l. 4. Discussion
Molar absorbance coefficients and reaction rate constants obtained are listed in Table 1. When the molar absorbance coefficients were determined, 1 mL of aqueous solution containing 2.25 mg of M" powder was employed. Simple calculation led to the estimation of molar concentrations for the dimer and monomer, 12.9 and 25.8 mM, respectively. The molar concentration of the dimer experimentally obtained at t = 0 was 6.1 mM as shown in Figure 2a. This might be due to the fact that the monomer and other products produced during stirring of the solution were removed by bubbling with Ar gas just before optical measurements. Figure 2a also shows that the decomposition of the dimer did not necessarily produce the monomer. This meant that the dimer decomposed not only into the monomer but also into other products. Makino et al. reported that these products contained terr-butylnitrosohydroxylamine, tert-butyl alcohol, and i~0butene.I~This fact made the reaction rate constants obtained uncertain. These products were recognized in the NMR spectrum only when recorded at high sensitivity (data not shown), but no NMR signals due to these products were observed just after Ar bubbling. Since we could not exclude these products from the solutions when the reaction rate constant between OH radicals and the monomer was measured, it was safely concluded that the reaction rate constant between OH radicals and the dimer was obtained with high accuracy, whereas the reaction rate constant between OH radicals and the monomer was determined with slight uncertainty. In the present study the molar absorbance coefficient of the dimer was determined not at 287 nm (maximum absorbance wavelength) but at 320 nm. An aqueous solution prepared by dissolving a few milligrams of MNP powder in 1 mL of H20 is generally used for spin-trapping experiments. This condition gives the solution unobservable, strong optical absorbance at 287 nm. Therefore, the use of optical absorbance at 320 nm is preferable to that at 287 nm for actual spin-trapping experiments. The molar absorbance coefficient at 287 nm, was estimated to be about 5700. Makino et a1.I4 and Rao and BhaskarI7 reported that the maximum absorbance of the dimer was observed at 287 nm in H20 and at 295 nm in diethyl ether, and that of the monomer at 662 nm in H20 and at 675 nm in diethyl ether. However, the molar absorbance coefficients of
Kuwabara et al.
14082 J. Phys. Chem., Vol. 99, No. 38, 1995
the dimer and monomer were measured only in the diethyl ehter 295 (cdlmer - ca. 8000 and E ~ & , , , = ca.20). The values obtained in H20 in the present study were slightly lower than those values. Because of the quite low absorbance of the monomer at 662 nm, slight errors were unavoidable in determination of the molar absorbance coefficient of the monomer. In the present study, the reactions of SCN' and (SCN)2'- with MNP itself were not included when the experimental data were analyzed by eqs 3-7. If the rate constants of these reactions are fast, they must be included in the analysis of the competition kinetics. Since the SCN' radical has maximum light absorbance at 330 nm,I8 and decays by reacting with SCN- to produce (SCN)2*-, the amounts of SCN' generated and the subsequent decay curves in the presence or absence of MNP were measured by monitoring the absorbance at 350 nm. Using the rate constant, 7.0 x lo9 M-' s-I, of the reaction between SCN' and SCN-,I9 the reaction rate constant between SCN' and MNP was roughly estimated to be around 1 x lo8 M-' s-I, though the data showed some scattering at various concentrations of MNP because of the low optical density of SCN'. (Details will be published elsewhere.) This means that the reaction of SCN' with SCN- occurs in preference to that of SCN' with MNP. The reaction of (SCN)2*- with MNP was also examined analyzing the decay rate of (SCN)2'- in the presence or absence of MNP by monitoring the optical absorbance at 475 nm. It was found that the decay of (SCN)2*- obeyed second-order kinetics with the rate constant of 1.5 x lo9M-' s-l. This value was in fair agreement with the values cited in the table presented by Neta et u Z . , ~ O indicating that the (SCN)2'- diminishes by reacting with (SCN)2*- itself. The presence of MNP had little effect on the decay kinetics (data not shown). This means that the rate constant of the reaction of (SCN)2'- with MNP is considerably low compared to that of (SCN)2'(SCN)2'-. From these results it was concluded that the present competition experiments were not largely affected by the reactions of SCN' and (SCN)?*- with MNP. As shown in Table 1, OH radicals reacted with the monomer faster than the dimer. This result was the opposite of that obtained from the reaction between hydrated electrons and MNP.I2 Hydrated electrons reacted with the dimer faster than the monomer. The azo-type structure in the dimer (eq 9) may relate to its high reactivity with hydrated electrons. On the other hand, the high reactivity of OH radicals with the dimer is thought to be brought about by the nitroso group (-N=O) of the monomer. The reaction rate constant between OH radicals and MNP has already been reported (4.0 x lo* M-' s-' by Bakalik et a1.,215.0 x lo9 M-' s-I by Greenstock,22 and 2.5 x lo9 M-' s-l by Madden and T a n i g ~ c h i ~ The ~ ) . values reported by Greenstock and Madden and Taniguchi were similar to that obtained for the reaction between OH radicals and the dimer (3.3 x lo9 M-' s-l). In monomer-dimer equilibrium of M N P , the dimer excited in a concentration higher than that of the monomer as shown in Figure 2. Therefore, the values they obtained are inferred to reflect by the reaction between OH radicals and the dimer rather than that between OH radicals and the monomer.
+
In the present study we could detennine the molar absorbance coefficients of the dimer and monomer of MNP. Since the amounts of dissolved MNP powder do not necessarily reflect the theoretically calculated molar concentrations, as described above, the molar absorbance coefficients will provide a method to simply and quickly estimate the concentrations of the dimer and monomer after an adequate amount of MNP powder is dissolved in an aqueous solution. Furthermore, we could separately determine the reaction rate constants between OH radicals and the dimer and OH radicals and the monomer. Of all spin traps, MNP is the most useful reagent when the radical structures trapped are identified. Another spin trap, 3,6dibromo-4-nitrosobenzensulfonic acid, is also known to be useful for radical assignment, but only the deuterated form of this agent is applicable for this. Therefore, the reaction rate constants determined in the present study will be useful for quantitative spin-trapping experiments using MNP.24 Acknowledgment. This work was supported by Grant-inAid from the Scientific Research Fund of the Ministry of Education, Science and Culture of Japan (No. 04680207) and Intemational Core System for the Basic Research Fund of the Science and Technology Agency of Japan. The authors are grateful to Drs. S . Shimokawa and K. Takahashi and Mrs. H. Tanida, K. Sato, and M. Kitaichi, Faculty of Engineering, Hokkaido University, for technical assistance. References and Notes (1) Finkelstein, E.; Rosen, G. M.; Rauckman, E. J. Archiv. Eiochem. Eiophys. 1980,200, 1. (2)Kuwabara, M.; Inanami, 0.;Endoh, D.; Sato, F. Biochemistry 1987, 26,2458. (3)Kuwabara, M.; Hiraoka, W.; Sato, F. Biochemistry 1989,28,9625. (4) Hiraoka, W.; Kuwabara, M.; Sato, F.; Matsuda, A.; Ueda, T. Nucleic Acids Res. 1990,18, 1217. ( 5 ) Kuwabara, M.; Ohshima, H.; Sato, F.; Ono, A,; Matsuda, A. Biochemistry 1994,32, 10599. (6)Inanami, 0.; Kuwabara, M.; Hayashi, M.; Yoshii, G.; Syuto, B.; Sato, F. lnt. J. Radiat. Eiol. 1986,49, 47. (7)Inanami, 0.; Sato, F.; Kuwabara, M. Mol. Biol. (Life Sci. Adv.) 1993,12,133. (8)Saprin, A.;Piette, L. H. Archiv. Biochem. Eiophys. 1977,180,480. (9)Rosen, G. M.; Rauckman, E. J. Proc. Natl. Acad. Sci. U.S.A. 1981, 78,1346. (10)Joshi, A.;Rustgi, S.; Riesz, P. lnt. J. Radiat. Eiol. 1976,30, 151. (11)Makino, K. J . Phys. Chem. 1980,84, 1012. (12)Kuwabara, M.; Hiraoka, W.; Sawamura, S . ; Katayama, M. J. Am. Chem. SOC. 1991,113,3995. (13)Riesz, P.;Rustgi, S . Radiat. Phys. Chem. 1979,13,21. (14)Makino, K.; Suzuki, N.; Moriya, F.; Rokushika, S.; Hatano, H. Radiat. Res. 1981,86,294. (15)Kondo, T.; Aikawa, M.; Sumiyoshi, T.; Katayama, M. J. Phys. Chem. 1980,84,2544. (16) Ellison, D.H.; Salmon, G. A.; Wilkinson, F. Proc. R. SOC.London, Ser. A 1972,328,23. (17) Bakalik, D.P.;Thomas, J. K. J. Phys. Chem. 1977,81,1905. (18) Greenstock, C.L.Astr. 61st Can. Chem. Congr. 1978,OR-5, 65. (19) Madden, K. P.;Taniguchi, H. J . Am. Chem. Soc. 1991,113,5541. (20) Kuwabara, M.;Hiraoka, W.; Inanami, 0.;Ohshima, H.; Sato, F.; Miyake, S . ; Sawamura, S.;Ono, A.; Matsuda, A. Magn. Reson. Med. 1995, 6, 126. JP950693J
