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Static Magnetic Field Effect on the Fremy’s Salt-Ascorbic Acid Chemical Reaction Studied by Continuous-Wave Electron Paramagnetic Resonance N. Catallo,*,† S. Colacicchi,† V. Carnicelli,‡ A. Di Giulio,‡ F. Lucari,§ and G. Gualtieri‡ INFM, Department of Health Sciences, UniVersity of L’Aquila, Via Vetoio, Coppito 2, 67100 L’Aquila, Italy, Department of Biomedical Sciences and Technologies, UniVersity of L’Aquila, Via Vetoio, Coppito 2, 67100 L’Aquila, Italy, and INFM, Department of Physics, UniVersity of L’Aquila, Via Vetoio, Coppito 1, 67100 L’Aquila, Italy ReceiVed: July 6, 2009; ReVised Manuscript ReceiVed: NoVember 15, 2009
Static magnetic field effect in the framework of the radial pair mechanism (RPM) theory was studied on the biologically significant chemical reaction between ascorbic acid and Fremy’s salt. The data indicate that the reaction rate depends on the applied magnetic field strength. The time scale of the studied reaction and the improved continuous-wave electron paramagnetic resonance system allowed for the first time the direct comparison of the amplitude differences between exposed and control samples in the strictly same boundary conditions. Until now the RPM was studied in a different time scale, focusing only on faster reactions by time-resolved techniques or by spectrophotometer measurement. The magnetic field effects presently measured can not be extended tout court to living systems; however the understanding of magnetic field sensitivity in basic chemical reaction in vitro could help clarifying the underlying basic step of interaction between magnetic fields and biological systems. 1. Introduction Magnetic field effects are known in biology for a long time. Some species of insects and fishes, for example, are able to navigate by using their geomagnetic field perception.1-3 The interaction mechanism by which even weak fields can influence complex biological systems is supposed to operate at a molecular level. However, about 40 years of research in this field has not provided significant results to explain and predict the effect of magnetic field on human health. A consistent interaction mechanism has not yet been identified, and the largest part of research is trying to explain only phenomenological observations.1 The radical pair mechanism (RPM) is a reliable method because it is based on a theoretical model, and it is supported by experimental evidence.1-23 It is applied almost exclusively to basic chemical reactions because of the need to avoid the influence of the many variables introduced by living being’s biology. According to RPM, the magnetic field acts on a pair of weakly coupled radicals during their approach and before the expected interaction. The radical pair can be either in singlet (S) or triplet (T) states, and in absence of a magnetic field, the energy levels are separated by the exchange interaction J(r). Under strict conditions concerning the magnetic properties and the lifetime of the pair, the magnetic field changes the electronic spin state of the pair, thus affecting the result of chemical interaction. The function J(r) is supposed to exponentially decreas with the radical distance.9 If it is of the same order of magnitude of the hyperfine interaction (HFI), a transition between S and T states may occur. This event is known as intersystem crossing (ISC). In organic radicals, which typically * To whom correspondence should be addressed. Phone: +393496469434. Fax: +390862319939. E-mail:
[email protected]. † Department of Health Sciences, University of L’Aquila. ‡ Department of Biomedical Sciences and Technologies, University of L’Aquila. § Department of Physics, University of L’Aquila.
contain light atoms, the HFI is small. Thus the S-T transition takes place as soon as the Zeeman splitting (gµBB/p) become different from zero, i.e., for very low magnetic field. In the present study, the effect of static magnetic fields higher than the geomagnetic field was investigated in the aqueous solution of ascorbic acid (AH2) and Fremy’s salt nitroxide (FS). Ascorbic acid is a vital food nutrient for humans, and in blood circulation it is the most effective antioxidant. The nitroxides, being free radicals, may be dangerous at a molecular level and they generally have the capability to interact with many metabolic processes.24 The chemical interaction is well depicted in the framework of the RPM theory, the radical pair being composed by the paramagnetic FS and by the intermediate radical of AH2, the ascorbyl (A-•). The latter comes from the AH2 loss of one electron and two protons.8 The formation of A-• correspond to a first rapid step, where a fraction of FS is suddenly reduced. After this step, which is not observable in the time scale of EPR measurement, the redox reaction goes on in presence of A-•, which once more transforms the paramagnetic FS in the EPR silent diamagnetic hydroxylamine.25 The two steps represent the formation and decay of the ascorbyl radical and can be outlined by the following reactions
AH- + (SO3K)2NO• f A-• + (SO3K)2NO- + H+
(1) A-• + (SO3K)2NO• f + A(SO3K)2NO-
(2)
where the semidehydroascorbate (AH-) reagent derives from the first AH2 deprotonation, while the A product, in reaction 2, is the result of the loss of two electrons and two protons by ascorbic acid.8
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The radical pair interaction takes place in the second step, where at the same time the reagent species AH- is formed again by the following dismutation reaction, thus altering the reagents/ products ratio
2A-• + H+ f AH- + A
(3)
So the ascorbyl can be continuously reformed during the reaction time, and as a consequence, the reducing agent appears to be effective, although the stoichiometric ratio of the reagents is unfavorable: 2.25 mM ascorbic acid toward 17 mM Fremy’s salt. This feature might also be responsible for the long-lasting reaction. The FS-A-• radical pair is reactive only in its singlet state, that is, the 25% of the total S and T states distribution. The recombination probability is changed only by ISC, which mostly converts the T states in S states in dependence of magnetic field intensity,5 thus giving the possibility to perceive the field presence to the reagents. The measurements were based on the direct comparison between the EPR kinetics of samples exposed or not to static magnetic field generated by Helmholtz coils. The experimental protocol of this parallel acquisition was used by Gualtieri et al.26 Through the use of an additional magnetic field gradient each EPR spectra embodied a 2-fold signal from the exposed and control samples, which were simultaneously placed in the resonant cavity. The acquisition system was improved and controlled in order to carefully account for the normalization of all the boundary conditions for the two samples. Great accuracy is required in the comparison between the two systems because the expected possible effects6,10,27 could be very low (∼1%). The interest in this research was also driven by the continuous-wave electron paramagnetic resonance (CWEPR) observation of a long-lasting chemically induced dynamic electron polarization (CIDEP) phenomenon at 350 mT during the redox of the same chemical solution.8 The aim of this work is to look into the basic chemical step of magnetic field effect to not consider the complex additional factors normally present in living organisms. 2. Materials and Methods 2.1. Chemicals. Potassium nitrosodisulfonate, (K2NO(SO3)2), or FS was from Aldrich Chimica (Milano, Italy). Ascorbic acid was from Sigma (St. Louis, MO) and other chemical compounds, reagent grade, were from Fluka AG (Buchs, Switzerland). All the solutions were made in bidistilled water. The FS stock solution (33 mM) was prepared by dissolving the appropriate amount of solid K2NO(SO3)2 in a saturated solution of K2CO3, according to the procedure described in Okazaki et al.28 It was stored and protected from air and light at 4 °C, and it was used for the period of a month.25 The AH2 stock solution (25 mM), which requires a daily preparation, was obtained by dissolving 44 mg of AH2 in 10 mL of phosphate buffer saline (PBS) (0.1 M) at pH ) 7.8 to mimic physiological condition. The final concentration of ascorbic acid (2.25 mM) was chosen to attain a mean kinetic duration of about two hours.25 The FS was mixed with ascorbic acid to a final concentration of 17 mM in PBS. The 2-mL final volume was sufficient to fill at least 10 capillary pairs. Pyrex tubes with a nominal volume of 50 µL ( 0.5% were used. 2.2. Experimental Protocol and Parallel Acquisition. EPR measurements were done setting the span on the central line of
Figure 1. EPR central line of the first FS acquisitions of the sample pairs in 6 mT during the kinetic decay (upper panel). The application of a magnetic field gradient attained the spatial resolution of the two samples in the cavity. Peak to peak amplitudes of the first double acquisitions of the exposed-control samples of the same kinetic (lower panel).
Fremy’s salt triplet because of the equivalence of the three lines amplitude.8 In this spectral window it was also possible to observe the weak ascorbyl signal in the final part of the kinetic reaction. The EPR signal of the nitroxide was observed during its long-lasting reduction caused by AH2. The mixing time of FS with reducing agent AH2 was considered as zero time for the kinetic process. The mixed solution was then immediately divided in two samples, one placed in the environmental field (control) and the other exposed to a static magnetic field generated by Helmholtz coils (exposed). The EPR data acquisition was based on the use of a stationary gradient applied on the main magnetic field direction. This system allows to discriminate the signal of two capillaries placed about 2 mm apart at the same time in the resonant cavity. Each couple of capillary was respectively filled with the exposed/ control solution, thus the resulting spectrum showed two lines placed side by side that belonged to different samples.26 As an example, a spectrum is shown in the upper panel of Figure 1. The parallel acquisition has the advantage to permit the comparison of the control and the exposed signal amplitudes in a completely equivalent experimental condition. The magnetic field values used in the exposition protocol were 0.5, 1.5, 4.0, and 6.0 mT. Other measurements were made at environmental field or at 350 mT by exposing the samples into the main EPR magnetic field. The CWEPR (X band) spectrometer operated under the following conditions: central magnetic field 350 mT, scan range 0.8 mT, sweep time 35 s, time constant 100 ms, modulation frequency 100 kHz, modulation amplitude 0.1 mT, microwave power source 280 mW, attenuation 10 dB. The samples were stored at controlled temperature. Once the two capillaries were inserted into the resonant cavity, they were exposed to a higher magnetic field due to the presence of the EPR magnet and to a higher temperature due to the heating of
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the microwave field. To disregard these altered conditions, the pair of samples were frequently substituted in the cavity and after each replacement only the first immediate acquisition was taken as significant for the data analysis (Figure 1). The time delay of a first spectral acquisition for each capillary pair is mainly due to EPR tuning time, so the samples experienced a different condition only for few minutes, a very small amount of time with respect to the whole kinetic duration (2 h). The alteration introduced by EPR measurement can thus be regarded as a reading procedure, whereas the overall reaction process took place outside of the EPR spectrometer under controlled conditions. Although in the kinetic acquisition more than one spectrum was acquired, only the first one of every pair was considered. The mean temperature inside the resonant cavity was 17.0 ( 0.5 °C. For exposition fields ranging from 0.5 mT to 6.0 mT the temperature of samples stored outside the cavity was strictly kept constant at 11.0 ( 0.1 °C, through a refrigeration system controlled by a thermocouple with sensitivity =50 µV/°C.26 For the exposures to environmental field and to the EPR field it was instead possible to control the temperature by a thermostatic bath (-50-200 ( 0.5 °C), set at the same temperature of the resonant cavity. The peak to peak amplitudes of FS signal from exposed and control sample were plotted vs time (Figure 1, lower panel). Each of these kinetics was normalized for the amplitude of its first spectrum to equalize small differences in the starting amplitude values. This procedure is adopted because a variation of AH2 concentration within the range of the experimental error may have an evident effect on the FS signal amplitude. As can be seen in the lower panel of Figure 1, the trend of the kinetic functions was approximately linear. Also the point to point difference between the amplitudes of the two signals from the parallel acquisition were calculated and reported versus time. Since the differences between exposed and control samples appeared to diverge during the kinetic trend, they were elaborated with a linear fit, whose slope was used to estimate the magnetic field effect. Many other experimental aspects may greatly influence the signal intensities and must be considered. The protocol regarding the strict temperature control of capillaries and the accurate capillary filling procedure were previously reported.26 2.3. Position Effect Optimization. Two other parameters involved in the parallel acquisition were taken into account: field gradient homogeneity and sample positioning in the resonant cavity. Thus, to make a correct comparison of the spatial resolved spectra, it was necessary to verify the preliminary equality of the two capillary signals. Tests on spatial homogeneity of gradient coils demonstrated that it is good enough to be negligible compared to the error in the EPR peak to peak amplitude which was evaluate as 2% (data not shown). On the other hand, position test made by comparison between signals of capillary samples filled with the same solution and placed in the two cavity location showed a significant intensity difference. The signal of the capillary placed in the position A was smaller than in position B, therefore an improvement in the positioning of the samples was necessary. To improve the reliability of sample positioning, two positioners were built especially for these experiments. They were polytetrafluoroethylene (PTFE, commercial name Teflon) short tubes closely inserted between the capillaries and the two quartz
Catallo et al.
Figure 2. FS peak-to-peak amplitudes at 350 mT obtained without (upper panel) and with (lower panel) positioners. The measures were taken by removing and replacing the capillaries 1 and 2 both in A and B positions, changing the capillary position and repeating the tuning procedure for each point. The evident coincidence of two points between signals from different position is only accidental.
sample holders. They had a semicircular larger head designed to hold them in place, and they enabled a guided insertion of the samples. The correct positioning of the samples was effective to minimizes the dielectric losses in the cavity and to eliminate small errors in the angle of insertion, which could give a different filling of the sensitive region of the resonant cavity. The result was useful to improve the reproducibility of the signal amplitudes, as shown in Figure 2. The amplitude differences induced by the different locations in the cavity were tested with two standard capillary samples filled with the same probe: FS in high concentration (17 mM), diluted in PBS at pH ) 7.8, to give a good signal/noise ratio. The measures were taken by removing and replacing the capillaries (1 and 2) both in A and B positions, changing the capillary position and repeating the tuning procedure for each point. The protocol is aimed to simulate the experimental condition present during a kinetic acquisition. This procedure was carried out with and without positioners and the spreading of amplitude values are shown in Figure 2. The average difference between A and B signals without positioners was significant (∼4%), since almost coincident values were expected. Besides, the fluctuations around a single set of amplitudes from A or B position were only about 1%. The positioners reduced significantly the variability of signal intensity. After their use the average distance between values from A and B positions was about 1%, and the fluctuations become negligible with a mean relative error of less than 0.1%. The residual constant difference between the two cavity location is probably due to the RF or modulation field distribution, and it can be simply treated as an offset. 3. Results 3.1. Magnetic Field Effect on Kinetic Rate. The comparison between exposed and control kinetics was obtained by plotting
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Figure 3. Signal amplitude differences of the capillary pairs and their linear fits in the least-squares sense for all static magnetic fields, carried out at pH ) 7.8 and AH2 ) 2.25 mM (red ) 350 mT, blue ) 6 mT, magenta ) 4 mT, green ) 1.5 mT, and light-blue ) 0.5 mT).
Figure 4. Angular coefficients vs static magnetic field of parallel acquisition at pH ) 7.8 and AH2 ) 2.25 mM. Angular coefficients are an estimate of field effect on kinetic rate by comparison to control. Their values and the relative expositions are tabulated in Table 1. Error bars in the plot are obtained from the best fit of data.
the amplitudes as well as their point to point differences. The sample exposed to magnetic field always showed a faster decay process than the control one, except for the case at 0.5 mT in which the reaction rate was slower and the differences from control were negatives. The measured effect was in any case continuous with the magnetic field value, it decreases from higher field values to about 1 mT, and when it is near zero (no exposed-control difference) it reverses with a small slowing down of the reaction. During the decay process the field selective effect continued to be active, so the spectral amplitudes become more and more different throughout the time. The experimental points were calculated from amplitude differences and the plot versus time of parallel kinetics conducted in five different static fields values at pH ) 7.8 and AH2 ) 2.25 mM are shown in Figure 3. Exposed samples always increased their divergence from control, and the amount of this increase is dependent on the magnetic field value. To quantitatively assess the field effect and to compare the different exposure values, it was necessary to consider a fixed time. The 100th minute after the zero time mixing was considered. Differences were greater than 10% for the exposure at 350 mT; the effect is about 10% for exposure at 6 mT, and it decreases until it become half (5%) at 1.5 mT. For the lowest exposure at 0.5 mT the reverse effect had an extent of about -2%. Linear fit of the data was presented to clarify the dependence of the differences in reaction rate on the magnetic field. The fits characterize the approximately linear growth of magnetic field selective effect, and the slope of the straight lines correspond to a greater effect in presence of a greater field. The data were fitted by imposing their passing by the origin of the axes because at the starting point t ) 0 there was no difference between exposed and control samples (see Figure 3). The dependence on magnetic field is visualized in Figure 4 as kinetics angular coefficients versus static magnetic field intensities. Their values and the relative exposures are reported in Table 1. 3.2. CIDEP. The potential presence of a CIDEP phenomenon was also investigated on the present system; it appeared as a change in the nitroxide spectral line from absorption (A) to emission (E) at the end of the kinetic reduction.5,8 According
TABLE 1: Angular Coefficient Depending on Applied Magnetic Field magnetic field (mT)
angular coefficient (µV/min)
3350 6 4 1.5 0.5
1.1 0.9 0.7 0.5 -0.1
to the RPM theory, it was ascribed to altered energy levels population due to the S-T-1 level crossing. In the studied solution the CIDEP was observed only for the magnetic field value of 350 mT and only on the exposed sample, while it was absent in the control one. Because of the strong dependence of reaction rate on the temperature parameter, this was accurately controlled by putting the control samples outside the cavity in a thermostatic bath set at the same cavity temperature (17.0 ( 0.5 °C). Figure 5 shows in the upper panel a whole parallel acquisition of kinetic decay of 17 mM FS with 2.0 mM AH2, acquired maintaining the exposed capillary in the EPR cavity and the control one in the environmental field. The lower panel reports the zoom of the last minutes of the reaction. The signal of the control sample during the first 100 min decreased more slowly than the exposed one and then dropped sharply, disappearing after 106 min. The signal of the exposed sample was instead visible in absorption mode up to 110th minute until it vanished and then reappeared in emission until the end of the reaction (120 min). The data were plotted by assigning positive values to A phase and negative values to E phase. In the inferior plot it can be noticed that while control amplitude was going to zero the exposed sample was assuming negative values. This is a qualitative assessment that CIDEP effect is directly caused by the magnetic field. 4. Discussion The differences between exposed and control kinetics showed that the reaction rate is dependent on the applied magnetic field strength. The increase in the slope of the linear fits is well correlated to the magnetic field value but not strictly proportional
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Catallo et al. other research about the products recombination in different radical pairs.6,19 To see if the effect were reproducible in different experimental conditions, some tests at different concentrations, different pHs, and at pulsed magnetic field were also done. These preliminary tests seem to confirm the results, but more evidence is needed. Furthermore, the occurrence of CIDEP effect observed at 350 mT8 suggests that the higher value of magnetic field made it possible the S-T-1 transition increasing the gap between the energy levels T+1 and T-1 by Zeeman effect, as expected from RPM theory. However, the field effect on FS-A-• pair described here should not be generalized to every radical pairs. The conditions needed by the field to affect the recombination rate of a radical pair are very restrictive in the RPM theory.16,27 The reaction of the pair must be spin selective, and the lifetime of the pair must be long enough to allow a coherent intersystem crossing, that is the field-dependent S/T transitions in a range between 100-1000 ns.1 Nevertheless if the lifetime of the pair is longer than 300 ns, the described process become ineffective because of the presence of the relaxation mechanism. In particular, the S-T conversion takes place in a time in the order of p/A (s), where A is the mean value of the hyperfine coupling constant in frequency unit.1 For the present case, as mentioned above, A ) 0.75 mT, so in p unit
Figure 5. Peak-to-peak amplitude of FS EPR signal, in a whole parallel acquisition with exposure field 350 mT (upper panel) and the zoom of its end (lower panel). On the lower plot the error bars were not reported in order to make the figure easier to read. The kinetic was obtained maintaining a capillary in the cavity and the other one in environmental field. Emission EPR lines appeared only in the case of exposed sample.
to it, in agreement with another work2 and with the RPM theory which predicts a magnetic field saturation effect below 100 mT.5 Indeed the angular coefficient of the 350 mT exposure was not much higher than the other values. During the kinetic process the magnetic field persists in increasing the difference from the control, and it is not straightforward to quantify the field effect, but it is necessary to report it for a fixed kinetic time and the 100th minute after solution mixing was considered. In the present case the maximum effect found was more than 10% although the theoretical prediction is almost 20%.1,10,27,14 Similar experimental results are reported in the scientific literature, but until now the RPM was studied in a different time scale focusing only on faster reactions by time-resolved techniques or spectrophotometric measurement.5,11,12,18,19,21,23,27,29-32 The time scale of the studied reaction and the improved EPR system allowed, for the first time, the direct comparison of the amplitude differences between exposed and control samples in the strictly same boundary conditions, with the further possibility to identify and follow radicals and intermediate products during the whole slow phase of the nitroxide reduction.8 For very low magnetic field (0.5 mT) the effect on reaction rate reverses the sign and the radical pair seems to experience the low field effect (LFE). This happens because degenerate zero-field states of the radical pair spin system are no longer degenerate in the presence of a weak magnetic field. As indicated by theoretical studies,2,6,27 the field strength is smaller than the mean value of the two radicals hyperfine coupling constant. For ascorbyl the AA-• is about 0.2 mT, while AFS ≈ 1.3 mT, so their mean value is A ) 0.75 mT. In this case the recombination probability attain a minimum because the ISC is suppressed by the dipolar interaction, and as a consequence, the reaction rate decreases.33 These results are in agreement with
1/A ) 1/(0.75 · 28.02 · 106Hz) ≈ 5 × 10-8s ) 50 ns (4) The spin correlation gives the spin selective redox reaction and the ISC occur in 50 ns, an appropriate time for the observation of magnetic field effect. 5. Conclusion The results support the hypothesis that in free radical reactions the magnetic field acts through RPM or LFE (valid only in 0.5 mT expositions) and that it is possible to measure the effect of magnetic field on biologically relevant chemical reaction. The observation of a long-lasting CIDEP effect was already reported,8 but in the present study it was compared to the effect found in the same reaction conducted entirely outside the X-band EPR field, with samples picked up only for a short measurement. The lack of CIDEP in the unexposed samples is a strong validation that the magnetic field effectively takes part to the spin dynamics of the studied radical pair. Nevertheless, the magnetic field effects demonstrated in the present paper on the pure chemical reaction between Fremy’s salt and ascorbic acid can not be extended tout court to living systems, because of the complexity of biological context due to the presence of compensation mechanisms, which are still poorly understood. Even if conclusions on health issues can not actually be expected, the understanding of magnetic field sensitivity in basic chemical reactions in vitro can help clarifying the underlying basic step of interaction between magnetic fields and biological systems. Further development are planned to overcome an intrinsic instrumental limit, which made it impossible to observe the former fast reaction phase with the EPR technique because of the delay introduced by tuning time. An exposition system, suitable to be inserted into commercial spectrophotometers, is under construction. This will allow the magnetic field sample expositions and concomitant spectrophotometric measurements during the first reaction phase.
Fremy’s Salt-Ascorbic Acid Chemical Reaction References and Notes (1) Brocklehurst, B.; McLauchlan, K. A. Int. J. Radiat. Biolog. 1996, 69, 3. (2) Brocklehurst, B. Chem. Soc. ReV. 2002, 31, 301. (3) Beason, R. C. Integ. Comput. Biol. 2005, 45, 565. (4) Grissom, C. B. Chem. ReV. 1995, 95, 3. (5) Hayashi, H. Introduction to dynamic spin chemistry: magnetic field effects upon chemical and biochemical reactions; World Scientific: Singapore, 2004. (6) Timmel, C. R.; Henbest, K. B. Phil. Trans. R. Soc. 2004, 362, 2573. (7) Woodward, J. R. Prog. React. Kinet. Mech. 2002, 27, 165. (8) Gualtieri, G.; Colacicchi, S.; Corvaja, C. J. Chem. Soc., Perkin Trans. 2 2002, 1917. (9) Nagakura, S.; Hayashi, H.; Azumi, T. Dynamic spin chemistry; J. Wiley & Sons: Tokyo, 1998. (10) Salikhov, K. M.; Molin, Yu. N.; Sagdeev, R. Z.; Buchachenko, I. Spin polarization and magnetic effects in radical reactions; Elsevier: Amsterdam, 1984. (11) Steiner, U. E.; Ulrich, T. Chem. ReV. 1989, 89, 51. (12) Boxer, S. G.; Chidsey, C. E. D.; Roelofs, M. G. Ann. ReV. Phys. Chem. 1983, 34, 389. (13) Hoff, A. J.; Lous, E. J.; Moehl, K. W.; Dijkman, J. A. Chem. Phys. Lett. 1985, 114, 39. (14) Schulten, K. AdV. Solid State Phys. 1982, 22, 61. (15) Brocklehurst, B. J. Chem. Soc. Faraday Trans. II 1976, 72, 1869. (16) Adair, K. R. Rep. Prog. Phys. 2000, 63, 415. (17) Adair, K. R. Bioelectromagnetics 1999, 20, 255. (18) Henbest, K. B.; Maeda, K.; Athanassiades, E.; Hore, P. J.; Timmerl, C. R. Chem. Phys. Lett. 2006, 421, 571.
J. Phys. Chem. A, Vol. 114, No. 2, 2010 783 (19) Rodgers, C. T.; Norman, S. A.; Henbest, K. B.; Timmel, C. R.; Hore, P. J. J. Am. Chem. Soc. 2007, 129, 6746. (20) Timmel, C. R.; Till, U.; Brocklehurst, B.; McLauchlan, K. A.; Hore, P. J. Bioengineering and biophysical aspect of electromagnetic fieldsCRC Press: Boca Raton, FL, 2006. (21) Henbest, K. B.; Kukura, P.; Rodgers, C. T.; Hore, P. J.; Timmel, C. R. J. Am. Chem. Soc. 2004, 124, 8102. (22) Till, U.; Timmel, C. R.; Brocklehurst, B.; Hore, P. J. Chem. Phys. Lett. 1998, 298, 7. (23) Woodward, J. R.; Jackson, R. J.; Timmel, C. R.; Hore, P. J.; McLauchlan, K. A. Chem. Phys. 1997, 272, 376. (24) Kocherginsky, N.; Swartz, H. M.; Sentjurc, M. Nitroxide spin labels: reactions in biology and chemistry; CRC Press: Boca Raton, FL, 1995. (25) Colacicchi, S.; Carnicelli, V.; Gualtieri, G.; Di Giulio, A. Res. Chem. Intermed. 2000, 26, 879. (26) Gualtieri, G.; Colacicchi, S.; Carnicelli, V.; Di Giulio, A. Biophys. Chem. 2005, 114, 149. (27) Timmel, C. R.; Till, U.; Brocklehurst, B.; McLauchlan, K. A.; Hore, P. J. Mol. Phys. 1998, 95, 71. (28) Okazaki, M.; Toriyama, K. J. Magn. Res. 1988, 79, 158. (29) Hoff, A. J. Quart. ReV. Biophys. 1981, 14, 599. (30) Sengupta, T.; Basu, S. Spectrochim. Acta, A 2004, 60, 1127. (31) Timmel, C. R.; Woodward, J. R.; Hore, P. J.; McLauchlan, K. A.; Stass, D. V. Meas. Sci. Technol. 2001, 12, 635. (32) Dzuba, S. A.; Proskuryakov, I. I.; Hulseboscha, R. J.; Boscha, M. K.; Gasta, P.; Hoff, A. J. Chem. Phys. Lett. 1996, 253, 361. (33) O’Dea, R.; Curtis, A. F.; Green, N. J. B.; Timmel, C. R.; Hore, P. J. J. Phys. Chem. 2005, 109, 869.
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