- Email: [email protected]
Enhancement of cytochrome oxidase activity in 60 Hz magnetic fields
Bioelectrochemistry and Bioenergetics 45 Ž1998. 253–259
Enhancement of cytochrome oxidase activity in 60 Hz magnetic fields M. Blank ) , L. Soo Department of Physiology and Cellular Biophysics, Columbia UniÕersity, 630 W. 168 Street New York, NY 10032, USA Received 29 September 1997; revised 2 February 1998; accepted 2 February 1998
Abstract 60 Hz magnetic fields accelerate the oxidation of cytochrome C, a reaction catalyzed by cytochrome oxidase, an electron transport enzyme of the mitochondrial redox chain. The effects of magnetic fields on this enzyme reaction are similar to effects on the Na,K-ATPase reaction. The acceleration due to the magnetic field, is inversely related to the basal enzyme rate. The greater the normal enzyme activity, the smaller the effect of an applied magnetic field. The acceleration varies with the magnetic field strength. The increase in the oxidation rate constant is 20–30% at field strengths below 3 mT, and a factor of about 2 between 6–10 mT. Data at low field strengths suggest that the threshold is below 0.5 mT, in the same range as thresholds for Na,K-ATPase function Ž0.3 mT. and stimulation of transcription Ž- 0.8 mT.. The mobile charge interaction ŽMCI. model, which proposes that electric and magnetic fields interact with moving charges as they would in any conductor, was based on studies of the Na,K-ATPase enzyme reaction. Similar results with cytochrome oxidase support the MCI model in the interaction of magnetic fields with electron transfer during this oxidation reaction. q 1998 Elsevier Science S.A. All rights reserved. Keywords: 60 Hz magnetic fields; Electromagnetic fields; Cytochrome oxidase; Enzyme kinetics; Redox reaction kinetics
1. Introduction 1.1. Na,K-ATPase studies An understanding of interaction mechanisms w1x is critical for assessing the health implications of biological effects of environmental electric and magnetic fields. The Na,K-ATPase has been a useful model for studies at the molecular level w2–9x, and we have defined thresholds w10,11x in both electric and magnetic fields. The data w4–11x are summarized in the diagram of Fig. 1, which shows that signal transduction, the transfer of energy from field to enzyme, depends ultimately on the basal enzyme activity. All experimental manipulations that result in a slow enzyme reaction rate, lead to a large positive response to either field. If the enzyme is at optimal activity, there is a smaller response, and the effects of electric and magnetic fields are in opposite directions. The dependence on basal enzyme activity indicates that reactions during physical transduction involve charge movements crucial to enzyme function. The calculated Lorentz force on a mov-
)
Corresponding author.
0302-4598r98r$19.00 q 1998 Elsevier Science S.A. All rights reserved. PII S 0 3 0 2 - 4 5 9 8 Ž 9 8 . 0 0 0 8 6 - 5
ing charge Žat assumed velocities based on the time scale of reaction rates. is negligibly small, but it is clear that the reaction rate is accelerated in a weak field. The relation to enzyme activity, and the change in sign of the electric field effect with increasing enzyme activity, have led to the mobile charge interaction ŽMCI. model, where it is assumed that the weak fields interact with moving charges as they would in any conductor, and that this interaction is sufficient to lead to interference with the reaction. To determine if this idea is general, we have looked for similar effects in other enzymes. Electric field effects, similar to those on the Na,KATPase, have already been shown w12x on the proton translocating F0 F1-ATPase of the mitochondrion. Under optimal conditions, there is inhibition of F0 F1-ATPase activity of Escherichia coli AN346 in electric fields. The inhibition increases with field strength, and is also a function of frequency in the range 0.01–10,000 Hz. Inhibition is minimal around 1 kHz at low field strength, and at about 30 Hz at higher field strength. Studies with the F0 F1-ATPase from mutant strains AN719 and AN781, with defects in the F0 a-subunit and the F1 e-subunit, respectively, showed lower frequency dependence around the minimum. Loss of frequency dependence was also
254
M. Blank, L. Soo r Bioelectrochemistry and Bioenergetics 45 (1998) 253–259
Fig. 1. Summary of results for many Na,K-ATPase preparations and experimental conditions Ždifferent temperatures, preparation ages, concentrations of the inhibitor ouabain. in response to electric and magnetic fields. Er A is the ratio of enzyme activity in an electric fieldrcontrol activity, and Mr A, is the ratio of enzyme activity in a magnetic fieldrcontrol activity. The summary graphs are shown on the same Na,K-ATPase activity scale. In the low activity range, the effects of the two fields are similar; at high activity, the effects are in opposite directions.
observed for precursor F0 F1 when activity decreased during cold storage. The frequency dependence of electric field inhibition appears to require the native enzyme structure. We have also tried to relate the effect of magnetic fields on an enzyme to a known charge flow. Studies with the Na,K-ATPase have not defined the nature of the charges, although the ability of electric double layer theory to account for reversal of the electric field effect with enzyme activity w7,11x suggests that the charges are mobile. There are charge movements w13x prior to the ionic fluxes associated with the ion pump, but it is hard to characterize the sign of the charge and, therefore, the direction. In the present research on cytochrome oxidase, a multi-component electron transport enzyme in the mitochondrial redox chain, both the charges Želectrons. and the direction of movement Žoxidation and reduction of cytochrome C . are known. 1.2. Cytochrome oxidase as a model Cytochrome oxidase has advantages as a model system for testing the role of mobile charges in the interaction of magnetic fields. It is the key enzyme at the end of the mitochondrial redox chain of aerobic metabolism, oxidizing cytochrome C and becoming reduced when combining with oxygen. Each cytochrome C transfers one electron to the enzyme; the reaction with oxygen requires an overall transfer of 4 electrons. The reaction can be studied spectrophotometrically, and the rate can be varied by controlling the concentrations of oxidized and reduced forms of cytochrome C. Cytochrome oxidase has 13 different subunits and a total molecular weight around 200,000 Da. Tsukihara et al. w14x have described the structure of oxi˚ resolution. dized cytochrome C oxidase at 2.8 A
Two previous studies have shown changes in the activity of cytochrome oxidase in magnetic fields. Gorczynska et al. w15x exposed the enzyme to large static fields Ž0.07–1.3 T. for up to 3 h, and reported changes in activity following exposure. Nossol et al. w16x also used large static fields Žin the range 0.3–10 mT., and reported significant changes under certain conditions. Of relevance to this study, Nossol et al. showed that 50 Hz Ž10 and 50 mT. magnetic fields accelerate steady state oxygen consumption. Using the terminology of enzyme kinetics, they reported results in terms of turnover numbers, and referred to high and low Žbinding. affinity states of cytochrome oxidase. Enzyme kinetics Žalso known as saturation kinetics. analysis applies only to the steady state. In this paper, we chose conditions where we could apply non-steady state kinetic analysis to give results in terms of reaction rate constants. We also exposed the enzyme to 60 Hz and much lower field strengths Žbelow 10 mT. than in the earlier studies. The focus on the initial oxidation of cytochrome C, without the many intermediate redox reactions within the enzyme complex, is much simpler than measuring the reaction with oxygen. A magnetic field can accelerate electron transfer associated with both oxidation and reduction, but by choosing to start with a fully reduced species, only oxidation is affected initially. The molecular Ždipolar. electric fields in cytochrome C and cytochrome oxidase have a strong effect on their orientation prior to binding and electron transfer w17x. It is not clear if these forces play a role when there are applied magnetic fields. There is no preferred orientation of the enzyme relative to the applied magnetic field, but the experimental results suggest that the electrical forces make it more likely for the magnetic field to accelerate rather than inhibit the transfer of electrons.
2. Methods 2.1. Magnetic field exposure conditions We have been using a unique exposure system w6,7x for studying the effects of separately controlled electric or magnetic fields, as well as the phase angle between them when they act simultaneously. The instrument was designed and constructed by Electric Research and Management, Pittsburgh, with the following specifications: frequency range: 1–3000 Hz, magnetic field: 0–1 mT, electric field: 0.2–200 mVrcm, phase between electric and magnetic fields: 0–3598. In this study, we have used 60 Hz 0–10 mT magnetic fields, which are uniform within 3% in the 5-cm cube volume used for exposure. This volume is kept at constant temperature with circulated water from a thermostat. Fred Dietrich ŽElectric Research and Management. and Martin Masakian ŽNIST. checked the equipment and found that it was performing within specifications, and that stray fields were below 0.1 mT.
M. Blank, L. Soo r Bioelectrochemistry and Bioenergetics 45 (1998) 253–259
2.2. Cytochrome oxidase preparation In our studies, cytochrome oxidase was prepared from livers of Sprague–Dawley rats. Livers were weighed, cut and homogenized in ice-cold isotonic sucrose Ž8.5 gr100 ml., and 120 ml of homogenate Ž1-g liver per 10-ml homogenate. were centrifuged for 15 min at 2000 rpm to sediment the nuclei and red blood cells. The supernatant was removed, and the sediment was washed by resuspension in 30-ml isotonic sucrose, and recentrifuged at 2000 rpm for 15 min. The supernatant and washings from the nuclear fractions were combined and centrifuged at 8500 rpm for 15 min to sediment the mitochondria. The supernatants were discarded, and the mitochondria were washed with isotonic sucrose, resuspended in isotonic sucrose and stored at y708C. The supernatant obtained after resedimentation of the mitochondria was retained. The final suspension of mitochondria was distinctly yellow in color. The protein concentrations and the enzymatic properties of different preparations are about the same, the concentrations being in the range of about 4-mg proteinrml. Cytochrome C ŽSigma C2506 from horse heart. solutions were prepared by dissolving 100 mg in 8.0 ml of a 0.01-M potassium phosphate pH 7.0 buffer. The cytochrome C was reduced by adding between 3 and 5 mg of L-ascorbic acid, sodium salt. By varying the amount of L-ascorbic acid Žsodium salt., we change the balance between reduced and oxidized species present initially, and therefore, the effective rate of oxidation. These solutions were used to study the effects of magnetic fields as a function of the net reaction rate. To determine the rate constant for oxidation, we removed excess ascorbate by dialyzing against 800 ml of a 0.01-M potassium phosphate buffer for about 7 h, at 0–48C, with two changes of buffer. The dialysis tubing ŽBaxter. had a molecular weight cutoff of 6000–8000. The dialysate, containing reduced cytochrome C, was brought up to a final volume of 10.0 ml with 0.01-M potassium phosphate pH 7.0 buffer. The cytochrome C remains reduced for several months if stored at 0–48C. 2.3. Cytochrome oxidase assay by spectrophotometric rate determination Enzyme activity was determined by the method of Smith w18x, where the rate of oxidation of reduced cytochrome C is measured spectrophotometrically by the decrease in optical density at 550 m m Žthe peak in the a-band of the absorption spectrum of cytochrome C ., as the reduced cytochrome is oxidized. Addition of K 3 FeŽCN. 6 solution is used to determine the optical density of completely oxidized cytochrome C at the end of an experiment. Sham controls are run with no applied field. The following reagents were prepared. ŽA. 0.1-M potassium phosphate buffer; pH 7.0 at 258C using monobasic potassium phosphate, adjusted to pH 7.0 with 1.0 M KOH.
255
ŽB. 0.01-M potassium phosphate buffer, pH 7.0 at 258C Žby diluting reagent A.. ŽC. 1% Žwrv. cytochrome C, reduced solution was prepared by dissolving 100 mg Ž8.07 = 10y3 mmol. of cytochrome C in 10 ml of Reagent B Ž8.07 = 10y4 M.. The cytochrome C was reduced by adding 4.0 mg of L-ascorbic acid, sodium salt Ž0.02 mmol.. ŽD. 0.1-M potassium ferricyanide solution used to prepare 10 ml of potassium ferricyanide in Reagent A. ŽE. 1% Žvrv. Tween 80 in 50 ml of Reagent A which contain 0.25 M of sucrose. This was the final solvent for the mitochondrial preparation. Details of the assay are as follows: in both control and magnetic field samples, each test tube contained 4.5 ml of Reagent B and 0.15 ml of Reagent E, and was placed in a water bath Ž248C.. At zero time, 0.15 ml of Reagent C Žor sometimes 0.20 ml, depending on the activity of the mitochondria. was added to start the reaction. At 1 min, and after each minute thereafter, 1.5 ml was withdrawn from the reacting solution, placed in a cuvette, and a spectrophotometer reading was taken at 550 nm Žin less than 15 s.. After the reading, the solution was immediately returned to the reaction mixture. This procedure was repeated for 8 min. After that, 0.1 ml of Reagent D was added to the reacting solution to complete the oxidation, and readings were continued at each minute 4 more times. The times used for the calculations are the times the sample was taken, which differs by 10–15 s from the time of the reading, but the same procedure was used for the controls and exposed samples. The Milton Roy Spectronic 401 spectrophotometer used for the determination of OD at 550 nm actually had a small magnetic field Žabout 1 mT. where the cuvette was placed for a reading. Since this is above the estimated threshold level, care was taken to minimize exposure to less than 15 s per reading, and the exposed volume was then returned to the reacting solution and thereby diluted. This minimal exposure was the same in both control and experimental groups. When studying the effects of magnetic fields, the data points are the average of at least four Žgenerally 5–8. determinations and four Žgenerally 5–8. controls, and the standard errors are around 5%. The rate data are reported as in the past as the ratio MrC, the value in Magnetic fieldrControl activity. Experiments, using cytochrome C preparations that initially contain only the reduced form, enable calculation of rate constants. These results will be described below. 2.4. Calculation of rate constants in dialyzed solutions In studying the Na,K-ATPase reaction, which proceeds by steady state enzyme kinetics, the data were reported in the form MrC. This is a standard way of reporting results, and data for cytochrome C oxidation will be reported in the same way. However, the cytochrome C oxidation
256
M. Blank, L. Soo r Bioelectrochemistry and Bioenergetics 45 (1998) 253–259
reaction is not in a steady state. The reaction is very fast at the beginning of the process, and falls off rapidly because of an appreciable back reaction. Although the kinetics of this rapidly equilibrating reaction are more complicated, measurements can be analyzed in terms of fundamental rate constants when excess reducing agent Žascorbate. is removed by dialysis. When this is done, concentrations of oxidized and reduced cytochrome C at any time can be determined from OD550 readings. Fig. 2 illustrates the basis of the calculation. If woxx s concentration of oxidized form, and wredx s concentration of reduced form, the initial reading Žat time s 0. corresponds to a fully reduced cytochrome C concentration, wredx, and the final reading, after addition of Reagent D, corresponds to fully oxidized cytochrome C concentration, woxx. Intermediate readings indicate the relative proportions of the two forms. The difference between the initial and final readings is the total concentration, woxx q wredx, and the difference between the initial reading and the reading at any time is woxx. The value of wredx is the difference between these two values. The net rate of change of reduced species in an equilibrating reaction, wredx l woxx, Rate s k o w red x y k r w ox x ,
Ž 1.
where k o is the rate constant for oxidation and k r is the rate constant for reduction. When the readings reach a plateau, the rate is zero, and k o w red x s k r w ox x .
Ž 2.
This occurs in our system at a ratio around 15:1 to 20:1 oxidized to reduced forms, and from this, one can calculate the equilibrium constant, K. When the ratio is 15:1, K s k ork r s 15,
Ž 3.
and the redox potential Žabout 69 mV..
Fig. 3. Dependence of the cytochrome oxidase reaction rate on the driving concentration, the concentration of fully reduced cytochrome C, divided by wredxywoxxr154. The correction factor takes into account the back reaction due to oxidized cytochrome C, and enables calculation of the correct value of the rate constant over the entire reaction. The rate constant is the slope of the line, according to Eq. Ž4..
When the data are plotted as woxx concentration versus time, the slope, which is equal to the rate of oxidation, can be determined at each time point. The rate constant, k o , is the proportionality constant between the rate of oxidation and the driving concentration Žwredx.. Because of the back reaction due to the buildup of woxx, the net oxidation rate must be corrected in determining the rate constant. Using the equilibrium constant in Eq. Ž3., Eq. Ž1. can be rewritten Rate s k o w red x y k o w ox x r15 s k o w red x y w ox x r15 4 .
Ž 4. The correction factor due to the back reaction is needed in order to calculate the correct rate constants ŽFig. 3.. When the correction for the back reaction is not made, the slope can differ by as much as 50%. Fig. 3 shows that when the oxidation reaction is treated as an equilibrating reaction, the rate constant Ži.e., the slope. is essentially constant over the whole time course.
3. Results
Fig. 2. Analysis of the cytochrome oxidase reaction, starting with a fully reduced cytochrome C preparation, where the reducing agent Žascorbate. has been removed by dialysis. By 8 min, there is an equilibrium mixture of oxidized and reduced forms. When an excess of ferricyanide is added, the cytochrome C becomes fully oxidized. The concentrations of oxidized Žwoxx. and reduced Žwredx. cytochrome C, shown at equilibrium, can be determined at any time from the OD550 Žoptical density at 550 m m ., as described in the text.
Our method determines the change in cytochrome C concentration as a function of time, based on the change in OD550 Žoptical density at 550 m m .. Spectrophotometer readings are essentially a linear concentration scale indicating the relative proportions of the oxidized and reduced forms of cytochrome C, as described in Fig. 2. Fig. 4 shows data for oxidation of cytochrome C under control conditions, and in the presence of a 10 mT field. Similar curves at other field strengths, were used to calculate MrC. Fig. 5, which shows MrC for four field strengths, indicates that the peak rate increases with field strength. In
M. Blank, L. Soo r Bioelectrochemistry and Bioenergetics 45 (1998) 253–259
257
Fig. 6. A plot of the ratio Mr C, where M is the rate in a magnetic field and C is the control vs. the magnetic field strength. The data points are the average of at least four runs, and standard errors are indicated. The threshold for response of cytochrome oxidase to magnetic fields can be estimated by extrapolating the measured reaction rates at different field strengths to zero effect. The least squares line Žslopes 0.00377, intercept s 0.979. show that the estimated threshold is below 10 mG Ž1 mT..
Fig. 4. Effect of a 100-mG Ž10 mT. field on cytochrome oxidase activity as a function of time. The data show the fraction of cytochrome C oxidized under control Žv . and magnetic field Žsquare filled with diagonal lines.. Data points Žaverage five determinations., and standard errors for the two curves are about "0.05.
all cases, the initial rate is very rapid, and then falls off with time. The dependence of MrC on field strength can be used to estimate the threshold for response of cytochrome oxidase to magnetic fields. An extrapolation of the measured reaction rates at low field strengths to zero effect would intersect the x-axis at the threshold. Fig. 6 shows the initial reaction rates Ždetermined at 1 min.. A least squares line with slope 0.00377 intersects the x-axis at 5.7 mG Ž0.57
Fig. 5. A plot of the normalized effect of a magnetic field, Mr C, where M are the data in a magnetic field and C are for the control. Data are shown for 20 mG Ž^., 50 mG ŽI., 70 mG Žcircle filled diagonal lines. and 100 mG ŽB.. The data points are the average of at least four runs, and standard errors at the 1-min point Žshown in Fig. 6. are about "0.05.
mT., estimating the threshold in this case well below 10 mG. Another important result that emerges from the kinetics data, and that is similar to earlier observations on Na,KATPase ŽFig. 1., is shown in Fig. 7. The magnitude of the magnetic field effect, MrC, depends upon the absolute reaction rate Žmeasured by the rate at the first minute.. The inverse correlation of the magnetic field effect with rate
Fig. 7. The dependence of the magnetic field effect, Mr C, on the initial reaction rate Žfraction cytochrome C oxidizedrmin.. The inverse correlation of Mr C with rate is similar to observations on Na,K-ATPase given in Fig. 1, where the rate is the enzyme activity.
258
M. Blank, L. Soo r Bioelectrochemistry and Bioenergetics 45 (1998) 253–259
4. Discussion 4.1. Magnetic fields affect electron transport in cytochrome oxidase function
Fig. 8. Variation of the rate constant of the cytochrome oxidase reaction with 60-Hz magnetic fields in the range 0–100 mG Ž0–10 mT., is shown for two dialyzed preparations. Standard errors of the points are in the range of 5–10%.
indicates that the magnetic field can be swamped by the endogenous activity. Up to this point, the data presented have been given in a format that can be compared to earlier results with Na,KATPase. In Figs. 8 and 9, we summarize measurements made on dialyzed solutions that enable calculation of reaction rate constants. Fig. 8 shows the variation of the rate constant of the cytochrome oxidase reaction at low magnetic field strengths for two dialyzed preparations. The magnetic field increases the rate constant by 20–30% at field strengths below 3 mT, and by a factor of 2–3 between 6–10 mT. These data can also be used to estimate the threshold for response of cytochrome oxidase to magnetic fields by extrapolating the measured reaction rates at low field strengths to zero effect. Fig. 9 shows that, by this method, the threshold appears to be below 0.5 mT Ž5 mG. ŽThe 1.8 point at 4 mG was obtained under the same conditions as the others, and we are unable to explain why the result did not fit the pattern...
From the results presented here, it appears that 60 Hz magnetic fields accelerate electron transport from cytochrome C to the enzyme, cytochrome oxidase. In experiments comparing the reaction rates with Ž M . and without Ž C . fields, the ratio MrC is greater than one. Under conditions where the reaction rate constant can be calculated, its value increases in a magnetic field. In our studies with the Na,K-ATPase, we found a similar effect of a magnetic field, but could not identify the charges affected. With cytochrome oxidase, the process affected is electron transport. In our experimental system, the total number of electrons transported by oxidation of 10y7 mol of cytochrome C in each reaction tube is about 10 17 electrons. The rate data of Fig. 7 enable us to estimate the electrons normally moved by the reaction, as well as the additional electrons moved by the field. When the control rate is very rapid, about 10 16 electronsrmg proteinrmin, there is no increase due to the field. When the control rate falls to about one third, the 30–40% increase due to the field is about 10 15 electronsrmg proteinrmin ŽIf all the protein were cytochrome oxidase, 1 mg protein would be 3 = 10 15 molecules... Just as found for the Na,K-ATPase, we observe that there is a large positive response to a magnetic field when cytochrome oxidase is not very active, and there is a smaller response at optimal activity. It appears that magnetic fields compete with the normal driving forces, and increase function only when the control level is relatively slow or below normal. This dependence on the biological state also seems to be true in therapeutic applications of magnetic fields w19x. It has always been considered odd that magnetic fields have no apparent effect on normal bones while they can accelerate the healing rate of broken bones. Like the magnetic field effect on the enzyme, therapeutic effects due to magnetic fields appear only when the tissue is damaged and its function diminished. 4.2. The mobile charge interaction (MCI) model
Fig. 9. Variation of the rate constant of the cytochrome oxidase reaction at low magnetic field strengths is shown for the two dialyzed preparations in Fig. 8. Standard errors of the points are in the range of 5–10%.
The mobile charge interaction ŽMCI. model proposes that magnetic fields interact with moving charges, as they would in any conductor, and that the observed dependence on basal enzyme activity is due to field interaction with charge movements during enzyme function. The effects reported here for cytochrome oxidase are similar to earlier results with Na,K-ATPase, and support the MCI interpretation of the data. These studies further show that the relation of signal transduction to enzyme activity applies to
M. Blank, L. Soo r Bioelectrochemistry and Bioenergetics 45 (1998) 253–259 Table 1 Electric and magnetic field thresholds
Na,K-ATPase Cytochrome C oxidase Biosynthesis ŽHL60 cells. Biosynthesis ŽSciara cells. Epidemiological threshold
259
Acknowledgements
Electric field
Magnetic field
0.5 mVrm y - 3 mVrm y
- 0.3 - 0.5 - 0.8 - 0.8 0.2
mT mT mT mT mT
the more complicated case of a non-steady state, equilibrating reaction, where there are simultaneous effects on the oxidation and reduction rate constants. In earlier studies, the qualitative difference between the effects of electric and magnetic fields lent support to the idea of mobile charges. The present studies with cytochrome oxidase also relate activity to the concentration of mobile charges, and show that a field effect is inversely proportional to the number of moving charges. The transient increase in oxidation rate, with no change in the equilibrium, means that oxidation must eventually slow down to normal as reduction speeds up to normal. The effect of the magnetic field is to accelerate the approach to equilibrium. 4.3. Thresholds for changes in cytochrome oxidase function in electric and magnetic fields To characterize biological responses to stimuli, it is customary to establish thresholds and effective ranges experimentally. In magnetic field studies, this process has been inhibited by theoreticians who claim that magnetic field effects are impossible at low field strengths. In view of the low thresholds that have now been found in several independent systems, with better than order of magnitude agreement in the results, this assertion no longer appears tenable. Table 1 shows data from our laboratory of a number of estimated thresholds for changes in enzyme activity stimulated by electric and magnetic fields. There are also data on biosynthesis showing the upper limits of thresholds. The values are consistent, and in the range of thresholds associated with disease in epidemiological studies. We have proposed that interaction of magnetic fields with moving charges could explain stimulation of transcription, where the magnetic field may interact with moving electrons within DNA w20x. Our current results suggest that the magnetic field would enhance charge flow, when the basal flow is relatively small, such as may occur when the cell is in a quiescent state or there is an obstruction Že.g., a kink. in the DNA conducting pathway.
We thank the Heineman Foundation and the NIEHS for support. References w1x M. Blank, ŽEd.., Electromagnetic Fields: Biological Interactions and Mechanisms. Advances in Chemistry, Vol. 250, American Chemical Society Press, Washington, DC, 1995. w2x E.H. Serpersu, T.Y. Tsong, Stimulation of a ouabain-sensitive Rbq uptake in human erythrocytes with an external electric field, J. Membr. Biol. 74 Ž1983. 191–201. w3x E.H. Serpersu, T.Y. Tsong, Activation of electrogenic Rbq transport of ŽNa,K.-ATPase by an electric field, J. Biol. Chem. 259 Ž1984. 7155–7162. w4x M. Blank, L. Soo, The effects of alternating currents on Na,KATPase function, Bioelectrochem. Bioenerg. 22 Ž1989. 313–322. w5x M. Blank, L. Soo, Temperature dependence of electric field effects on the Na,K-ATPase, Bioelectrochem. Bioenerg. 28 Ž1992. 291–299. w6x M. Blank, L. Soo, V. Papstein, Effects of low frequency electromagnetic fields on Na,K-ATPase activity, Bioelectrochem. Bioenerg. 38 Ž1995. 267–273. w7x M. Blank, L. Soo, Frequency dependence of Na,K-ATPase function in magnetic fields, Bioelectrochem. Bioenerg. 42 Ž1997. 231–234. w8x M. Blank, Na,K-ATPase function in alternating electric fields, FASEB J. 6 Ž1992. 2434–2438. w9x M. Blank, Electric and magnetic field signal transduction in the membrane Na,K-ATPase, Adv. Chem. 250 Ž1995. 339–348. w10x M. Blank, L. Soo, The threshold for alternating current inhibition of the Na,K-ATPase, Bioelectromagnetics 13 Ž1992. 329–333. w11x M. Blank, L. Soo, Threshold for Na,K-ATPase stimulation by EM fields, Bioelectrochem. Bioenerg. 40 Ž1996. 63–65. w12x S. Martirosov, M. Blank, Inhibition of F0 F1 -atpase activity in ACfields, Bioelectrochem. Bioenerg. 37 Ž1995. 153–156. w13x D.W. Hilgemann, Channel-like function of the Na,K pump probed at microsecond resolution in giant membrane patches, Science 263 Ž1994. 1429–1431. w14x T. Tsukihara, H. Aoyama, E. Yamashita, T. Tomizaka, H. Yamaguchi, K. Shinzawa-Itoh, R. Nakashima, R. Yaono, S. Yoshikawa, The whole structure of the 13-subunit oxidized cytochrome C oxi˚ Science 272 Ž1996. 1136–1144. dase at 2.8 A, w15x E. Gorczynska, G. Galka, R. Krolikowska, R. Wegrzynowicz, Effect of magnetic field on activity of cytochrome oxidase not moved or moved relative to magnetic field lines, Physiol. Chem. Phys. 14 Ž1982. 201–207. w16x B. Nossol, G. Buse, J. Silny, Influence of weak static and 50 Hz magnetic fields on the redox activity of cytochrome-C oxidase, Bioelectromagnetics 14 Ž1993. 361–372. w17x W.H. Koppenol, C.A.J. Vroonland, R. Braams, The electric potential field around cytochrome C and the effect of ionic strength on reaction rates of horse cytochrome C, Biochim. Biophys. Acta 503 Ž1978. 499–508. w18x L. Smith, Spectrophotometric assay of cytochrome C oxidase, Methods Biochem. Anal. 2 Ž1955. 427–434. w19x C.A.L. Bassett, Bioelectromagnetics in the service of medicine, in: M. Blank, ŽEd.., Electromagnetic Fields: Biological Interactions and Mechanisms, Advances in Chemistry, Vol. 250, 1995, 261–275. w20x M. Blank, R. Goodman, Do electromagnetic fields interact directly with DNA?, Bioelectromagnetics 18 Ž1996. 111–115.
Enhancement of cytochrome oxidase activity in 60 Hz magnetic fields M. Blank ) , L. Soo Department of Physiology and Cellular Biophysics, Columbia UniÕersity, 630 W. 168 Street New York, NY 10032, USA Received 29 September 1997; revised 2 February 1998; accepted 2 February 1998
Abstract 60 Hz magnetic fields accelerate the oxidation of cytochrome C, a reaction catalyzed by cytochrome oxidase, an electron transport enzyme of the mitochondrial redox chain. The effects of magnetic fields on this enzyme reaction are similar to effects on the Na,K-ATPase reaction. The acceleration due to the magnetic field, is inversely related to the basal enzyme rate. The greater the normal enzyme activity, the smaller the effect of an applied magnetic field. The acceleration varies with the magnetic field strength. The increase in the oxidation rate constant is 20–30% at field strengths below 3 mT, and a factor of about 2 between 6–10 mT. Data at low field strengths suggest that the threshold is below 0.5 mT, in the same range as thresholds for Na,K-ATPase function Ž0.3 mT. and stimulation of transcription Ž- 0.8 mT.. The mobile charge interaction ŽMCI. model, which proposes that electric and magnetic fields interact with moving charges as they would in any conductor, was based on studies of the Na,K-ATPase enzyme reaction. Similar results with cytochrome oxidase support the MCI model in the interaction of magnetic fields with electron transfer during this oxidation reaction. q 1998 Elsevier Science S.A. All rights reserved. Keywords: 60 Hz magnetic fields; Electromagnetic fields; Cytochrome oxidase; Enzyme kinetics; Redox reaction kinetics
1. Introduction 1.1. Na,K-ATPase studies An understanding of interaction mechanisms w1x is critical for assessing the health implications of biological effects of environmental electric and magnetic fields. The Na,K-ATPase has been a useful model for studies at the molecular level w2–9x, and we have defined thresholds w10,11x in both electric and magnetic fields. The data w4–11x are summarized in the diagram of Fig. 1, which shows that signal transduction, the transfer of energy from field to enzyme, depends ultimately on the basal enzyme activity. All experimental manipulations that result in a slow enzyme reaction rate, lead to a large positive response to either field. If the enzyme is at optimal activity, there is a smaller response, and the effects of electric and magnetic fields are in opposite directions. The dependence on basal enzyme activity indicates that reactions during physical transduction involve charge movements crucial to enzyme function. The calculated Lorentz force on a mov-
)
Corresponding author.
0302-4598r98r$19.00 q 1998 Elsevier Science S.A. All rights reserved. PII S 0 3 0 2 - 4 5 9 8 Ž 9 8 . 0 0 0 8 6 - 5
ing charge Žat assumed velocities based on the time scale of reaction rates. is negligibly small, but it is clear that the reaction rate is accelerated in a weak field. The relation to enzyme activity, and the change in sign of the electric field effect with increasing enzyme activity, have led to the mobile charge interaction ŽMCI. model, where it is assumed that the weak fields interact with moving charges as they would in any conductor, and that this interaction is sufficient to lead to interference with the reaction. To determine if this idea is general, we have looked for similar effects in other enzymes. Electric field effects, similar to those on the Na,KATPase, have already been shown w12x on the proton translocating F0 F1-ATPase of the mitochondrion. Under optimal conditions, there is inhibition of F0 F1-ATPase activity of Escherichia coli AN346 in electric fields. The inhibition increases with field strength, and is also a function of frequency in the range 0.01–10,000 Hz. Inhibition is minimal around 1 kHz at low field strength, and at about 30 Hz at higher field strength. Studies with the F0 F1-ATPase from mutant strains AN719 and AN781, with defects in the F0 a-subunit and the F1 e-subunit, respectively, showed lower frequency dependence around the minimum. Loss of frequency dependence was also
254
M. Blank, L. Soo r Bioelectrochemistry and Bioenergetics 45 (1998) 253–259
Fig. 1. Summary of results for many Na,K-ATPase preparations and experimental conditions Ždifferent temperatures, preparation ages, concentrations of the inhibitor ouabain. in response to electric and magnetic fields. Er A is the ratio of enzyme activity in an electric fieldrcontrol activity, and Mr A, is the ratio of enzyme activity in a magnetic fieldrcontrol activity. The summary graphs are shown on the same Na,K-ATPase activity scale. In the low activity range, the effects of the two fields are similar; at high activity, the effects are in opposite directions.
observed for precursor F0 F1 when activity decreased during cold storage. The frequency dependence of electric field inhibition appears to require the native enzyme structure. We have also tried to relate the effect of magnetic fields on an enzyme to a known charge flow. Studies with the Na,K-ATPase have not defined the nature of the charges, although the ability of electric double layer theory to account for reversal of the electric field effect with enzyme activity w7,11x suggests that the charges are mobile. There are charge movements w13x prior to the ionic fluxes associated with the ion pump, but it is hard to characterize the sign of the charge and, therefore, the direction. In the present research on cytochrome oxidase, a multi-component electron transport enzyme in the mitochondrial redox chain, both the charges Želectrons. and the direction of movement Žoxidation and reduction of cytochrome C . are known. 1.2. Cytochrome oxidase as a model Cytochrome oxidase has advantages as a model system for testing the role of mobile charges in the interaction of magnetic fields. It is the key enzyme at the end of the mitochondrial redox chain of aerobic metabolism, oxidizing cytochrome C and becoming reduced when combining with oxygen. Each cytochrome C transfers one electron to the enzyme; the reaction with oxygen requires an overall transfer of 4 electrons. The reaction can be studied spectrophotometrically, and the rate can be varied by controlling the concentrations of oxidized and reduced forms of cytochrome C. Cytochrome oxidase has 13 different subunits and a total molecular weight around 200,000 Da. Tsukihara et al. w14x have described the structure of oxi˚ resolution. dized cytochrome C oxidase at 2.8 A
Two previous studies have shown changes in the activity of cytochrome oxidase in magnetic fields. Gorczynska et al. w15x exposed the enzyme to large static fields Ž0.07–1.3 T. for up to 3 h, and reported changes in activity following exposure. Nossol et al. w16x also used large static fields Žin the range 0.3–10 mT., and reported significant changes under certain conditions. Of relevance to this study, Nossol et al. showed that 50 Hz Ž10 and 50 mT. magnetic fields accelerate steady state oxygen consumption. Using the terminology of enzyme kinetics, they reported results in terms of turnover numbers, and referred to high and low Žbinding. affinity states of cytochrome oxidase. Enzyme kinetics Žalso known as saturation kinetics. analysis applies only to the steady state. In this paper, we chose conditions where we could apply non-steady state kinetic analysis to give results in terms of reaction rate constants. We also exposed the enzyme to 60 Hz and much lower field strengths Žbelow 10 mT. than in the earlier studies. The focus on the initial oxidation of cytochrome C, without the many intermediate redox reactions within the enzyme complex, is much simpler than measuring the reaction with oxygen. A magnetic field can accelerate electron transfer associated with both oxidation and reduction, but by choosing to start with a fully reduced species, only oxidation is affected initially. The molecular Ždipolar. electric fields in cytochrome C and cytochrome oxidase have a strong effect on their orientation prior to binding and electron transfer w17x. It is not clear if these forces play a role when there are applied magnetic fields. There is no preferred orientation of the enzyme relative to the applied magnetic field, but the experimental results suggest that the electrical forces make it more likely for the magnetic field to accelerate rather than inhibit the transfer of electrons.
2. Methods 2.1. Magnetic field exposure conditions We have been using a unique exposure system w6,7x for studying the effects of separately controlled electric or magnetic fields, as well as the phase angle between them when they act simultaneously. The instrument was designed and constructed by Electric Research and Management, Pittsburgh, with the following specifications: frequency range: 1–3000 Hz, magnetic field: 0–1 mT, electric field: 0.2–200 mVrcm, phase between electric and magnetic fields: 0–3598. In this study, we have used 60 Hz 0–10 mT magnetic fields, which are uniform within 3% in the 5-cm cube volume used for exposure. This volume is kept at constant temperature with circulated water from a thermostat. Fred Dietrich ŽElectric Research and Management. and Martin Masakian ŽNIST. checked the equipment and found that it was performing within specifications, and that stray fields were below 0.1 mT.
M. Blank, L. Soo r Bioelectrochemistry and Bioenergetics 45 (1998) 253–259
2.2. Cytochrome oxidase preparation In our studies, cytochrome oxidase was prepared from livers of Sprague–Dawley rats. Livers were weighed, cut and homogenized in ice-cold isotonic sucrose Ž8.5 gr100 ml., and 120 ml of homogenate Ž1-g liver per 10-ml homogenate. were centrifuged for 15 min at 2000 rpm to sediment the nuclei and red blood cells. The supernatant was removed, and the sediment was washed by resuspension in 30-ml isotonic sucrose, and recentrifuged at 2000 rpm for 15 min. The supernatant and washings from the nuclear fractions were combined and centrifuged at 8500 rpm for 15 min to sediment the mitochondria. The supernatants were discarded, and the mitochondria were washed with isotonic sucrose, resuspended in isotonic sucrose and stored at y708C. The supernatant obtained after resedimentation of the mitochondria was retained. The final suspension of mitochondria was distinctly yellow in color. The protein concentrations and the enzymatic properties of different preparations are about the same, the concentrations being in the range of about 4-mg proteinrml. Cytochrome C ŽSigma C2506 from horse heart. solutions were prepared by dissolving 100 mg in 8.0 ml of a 0.01-M potassium phosphate pH 7.0 buffer. The cytochrome C was reduced by adding between 3 and 5 mg of L-ascorbic acid, sodium salt. By varying the amount of L-ascorbic acid Žsodium salt., we change the balance between reduced and oxidized species present initially, and therefore, the effective rate of oxidation. These solutions were used to study the effects of magnetic fields as a function of the net reaction rate. To determine the rate constant for oxidation, we removed excess ascorbate by dialyzing against 800 ml of a 0.01-M potassium phosphate buffer for about 7 h, at 0–48C, with two changes of buffer. The dialysis tubing ŽBaxter. had a molecular weight cutoff of 6000–8000. The dialysate, containing reduced cytochrome C, was brought up to a final volume of 10.0 ml with 0.01-M potassium phosphate pH 7.0 buffer. The cytochrome C remains reduced for several months if stored at 0–48C. 2.3. Cytochrome oxidase assay by spectrophotometric rate determination Enzyme activity was determined by the method of Smith w18x, where the rate of oxidation of reduced cytochrome C is measured spectrophotometrically by the decrease in optical density at 550 m m Žthe peak in the a-band of the absorption spectrum of cytochrome C ., as the reduced cytochrome is oxidized. Addition of K 3 FeŽCN. 6 solution is used to determine the optical density of completely oxidized cytochrome C at the end of an experiment. Sham controls are run with no applied field. The following reagents were prepared. ŽA. 0.1-M potassium phosphate buffer; pH 7.0 at 258C using monobasic potassium phosphate, adjusted to pH 7.0 with 1.0 M KOH.
255
ŽB. 0.01-M potassium phosphate buffer, pH 7.0 at 258C Žby diluting reagent A.. ŽC. 1% Žwrv. cytochrome C, reduced solution was prepared by dissolving 100 mg Ž8.07 = 10y3 mmol. of cytochrome C in 10 ml of Reagent B Ž8.07 = 10y4 M.. The cytochrome C was reduced by adding 4.0 mg of L-ascorbic acid, sodium salt Ž0.02 mmol.. ŽD. 0.1-M potassium ferricyanide solution used to prepare 10 ml of potassium ferricyanide in Reagent A. ŽE. 1% Žvrv. Tween 80 in 50 ml of Reagent A which contain 0.25 M of sucrose. This was the final solvent for the mitochondrial preparation. Details of the assay are as follows: in both control and magnetic field samples, each test tube contained 4.5 ml of Reagent B and 0.15 ml of Reagent E, and was placed in a water bath Ž248C.. At zero time, 0.15 ml of Reagent C Žor sometimes 0.20 ml, depending on the activity of the mitochondria. was added to start the reaction. At 1 min, and after each minute thereafter, 1.5 ml was withdrawn from the reacting solution, placed in a cuvette, and a spectrophotometer reading was taken at 550 nm Žin less than 15 s.. After the reading, the solution was immediately returned to the reaction mixture. This procedure was repeated for 8 min. After that, 0.1 ml of Reagent D was added to the reacting solution to complete the oxidation, and readings were continued at each minute 4 more times. The times used for the calculations are the times the sample was taken, which differs by 10–15 s from the time of the reading, but the same procedure was used for the controls and exposed samples. The Milton Roy Spectronic 401 spectrophotometer used for the determination of OD at 550 nm actually had a small magnetic field Žabout 1 mT. where the cuvette was placed for a reading. Since this is above the estimated threshold level, care was taken to minimize exposure to less than 15 s per reading, and the exposed volume was then returned to the reacting solution and thereby diluted. This minimal exposure was the same in both control and experimental groups. When studying the effects of magnetic fields, the data points are the average of at least four Žgenerally 5–8. determinations and four Žgenerally 5–8. controls, and the standard errors are around 5%. The rate data are reported as in the past as the ratio MrC, the value in Magnetic fieldrControl activity. Experiments, using cytochrome C preparations that initially contain only the reduced form, enable calculation of rate constants. These results will be described below. 2.4. Calculation of rate constants in dialyzed solutions In studying the Na,K-ATPase reaction, which proceeds by steady state enzyme kinetics, the data were reported in the form MrC. This is a standard way of reporting results, and data for cytochrome C oxidation will be reported in the same way. However, the cytochrome C oxidation
256
M. Blank, L. Soo r Bioelectrochemistry and Bioenergetics 45 (1998) 253–259
reaction is not in a steady state. The reaction is very fast at the beginning of the process, and falls off rapidly because of an appreciable back reaction. Although the kinetics of this rapidly equilibrating reaction are more complicated, measurements can be analyzed in terms of fundamental rate constants when excess reducing agent Žascorbate. is removed by dialysis. When this is done, concentrations of oxidized and reduced cytochrome C at any time can be determined from OD550 readings. Fig. 2 illustrates the basis of the calculation. If woxx s concentration of oxidized form, and wredx s concentration of reduced form, the initial reading Žat time s 0. corresponds to a fully reduced cytochrome C concentration, wredx, and the final reading, after addition of Reagent D, corresponds to fully oxidized cytochrome C concentration, woxx. Intermediate readings indicate the relative proportions of the two forms. The difference between the initial and final readings is the total concentration, woxx q wredx, and the difference between the initial reading and the reading at any time is woxx. The value of wredx is the difference between these two values. The net rate of change of reduced species in an equilibrating reaction, wredx l woxx, Rate s k o w red x y k r w ox x ,
Ž 1.
where k o is the rate constant for oxidation and k r is the rate constant for reduction. When the readings reach a plateau, the rate is zero, and k o w red x s k r w ox x .
Ž 2.
This occurs in our system at a ratio around 15:1 to 20:1 oxidized to reduced forms, and from this, one can calculate the equilibrium constant, K. When the ratio is 15:1, K s k ork r s 15,
Ž 3.
and the redox potential Žabout 69 mV..
Fig. 3. Dependence of the cytochrome oxidase reaction rate on the driving concentration, the concentration of fully reduced cytochrome C, divided by wredxywoxxr154. The correction factor takes into account the back reaction due to oxidized cytochrome C, and enables calculation of the correct value of the rate constant over the entire reaction. The rate constant is the slope of the line, according to Eq. Ž4..
When the data are plotted as woxx concentration versus time, the slope, which is equal to the rate of oxidation, can be determined at each time point. The rate constant, k o , is the proportionality constant between the rate of oxidation and the driving concentration Žwredx.. Because of the back reaction due to the buildup of woxx, the net oxidation rate must be corrected in determining the rate constant. Using the equilibrium constant in Eq. Ž3., Eq. Ž1. can be rewritten Rate s k o w red x y k o w ox x r15 s k o w red x y w ox x r15 4 .
Ž 4. The correction factor due to the back reaction is needed in order to calculate the correct rate constants ŽFig. 3.. When the correction for the back reaction is not made, the slope can differ by as much as 50%. Fig. 3 shows that when the oxidation reaction is treated as an equilibrating reaction, the rate constant Ži.e., the slope. is essentially constant over the whole time course.
3. Results
Fig. 2. Analysis of the cytochrome oxidase reaction, starting with a fully reduced cytochrome C preparation, where the reducing agent Žascorbate. has been removed by dialysis. By 8 min, there is an equilibrium mixture of oxidized and reduced forms. When an excess of ferricyanide is added, the cytochrome C becomes fully oxidized. The concentrations of oxidized Žwoxx. and reduced Žwredx. cytochrome C, shown at equilibrium, can be determined at any time from the OD550 Žoptical density at 550 m m ., as described in the text.
Our method determines the change in cytochrome C concentration as a function of time, based on the change in OD550 Žoptical density at 550 m m .. Spectrophotometer readings are essentially a linear concentration scale indicating the relative proportions of the oxidized and reduced forms of cytochrome C, as described in Fig. 2. Fig. 4 shows data for oxidation of cytochrome C under control conditions, and in the presence of a 10 mT field. Similar curves at other field strengths, were used to calculate MrC. Fig. 5, which shows MrC for four field strengths, indicates that the peak rate increases with field strength. In
M. Blank, L. Soo r Bioelectrochemistry and Bioenergetics 45 (1998) 253–259
257
Fig. 6. A plot of the ratio Mr C, where M is the rate in a magnetic field and C is the control vs. the magnetic field strength. The data points are the average of at least four runs, and standard errors are indicated. The threshold for response of cytochrome oxidase to magnetic fields can be estimated by extrapolating the measured reaction rates at different field strengths to zero effect. The least squares line Žslopes 0.00377, intercept s 0.979. show that the estimated threshold is below 10 mG Ž1 mT..
Fig. 4. Effect of a 100-mG Ž10 mT. field on cytochrome oxidase activity as a function of time. The data show the fraction of cytochrome C oxidized under control Žv . and magnetic field Žsquare filled with diagonal lines.. Data points Žaverage five determinations., and standard errors for the two curves are about "0.05.
all cases, the initial rate is very rapid, and then falls off with time. The dependence of MrC on field strength can be used to estimate the threshold for response of cytochrome oxidase to magnetic fields. An extrapolation of the measured reaction rates at low field strengths to zero effect would intersect the x-axis at the threshold. Fig. 6 shows the initial reaction rates Ždetermined at 1 min.. A least squares line with slope 0.00377 intersects the x-axis at 5.7 mG Ž0.57
Fig. 5. A plot of the normalized effect of a magnetic field, Mr C, where M are the data in a magnetic field and C are for the control. Data are shown for 20 mG Ž^., 50 mG ŽI., 70 mG Žcircle filled diagonal lines. and 100 mG ŽB.. The data points are the average of at least four runs, and standard errors at the 1-min point Žshown in Fig. 6. are about "0.05.
mT., estimating the threshold in this case well below 10 mG. Another important result that emerges from the kinetics data, and that is similar to earlier observations on Na,KATPase ŽFig. 1., is shown in Fig. 7. The magnitude of the magnetic field effect, MrC, depends upon the absolute reaction rate Žmeasured by the rate at the first minute.. The inverse correlation of the magnetic field effect with rate
Fig. 7. The dependence of the magnetic field effect, Mr C, on the initial reaction rate Žfraction cytochrome C oxidizedrmin.. The inverse correlation of Mr C with rate is similar to observations on Na,K-ATPase given in Fig. 1, where the rate is the enzyme activity.
258
M. Blank, L. Soo r Bioelectrochemistry and Bioenergetics 45 (1998) 253–259
4. Discussion 4.1. Magnetic fields affect electron transport in cytochrome oxidase function
Fig. 8. Variation of the rate constant of the cytochrome oxidase reaction with 60-Hz magnetic fields in the range 0–100 mG Ž0–10 mT., is shown for two dialyzed preparations. Standard errors of the points are in the range of 5–10%.
indicates that the magnetic field can be swamped by the endogenous activity. Up to this point, the data presented have been given in a format that can be compared to earlier results with Na,KATPase. In Figs. 8 and 9, we summarize measurements made on dialyzed solutions that enable calculation of reaction rate constants. Fig. 8 shows the variation of the rate constant of the cytochrome oxidase reaction at low magnetic field strengths for two dialyzed preparations. The magnetic field increases the rate constant by 20–30% at field strengths below 3 mT, and by a factor of 2–3 between 6–10 mT. These data can also be used to estimate the threshold for response of cytochrome oxidase to magnetic fields by extrapolating the measured reaction rates at low field strengths to zero effect. Fig. 9 shows that, by this method, the threshold appears to be below 0.5 mT Ž5 mG. ŽThe 1.8 point at 4 mG was obtained under the same conditions as the others, and we are unable to explain why the result did not fit the pattern...
From the results presented here, it appears that 60 Hz magnetic fields accelerate electron transport from cytochrome C to the enzyme, cytochrome oxidase. In experiments comparing the reaction rates with Ž M . and without Ž C . fields, the ratio MrC is greater than one. Under conditions where the reaction rate constant can be calculated, its value increases in a magnetic field. In our studies with the Na,K-ATPase, we found a similar effect of a magnetic field, but could not identify the charges affected. With cytochrome oxidase, the process affected is electron transport. In our experimental system, the total number of electrons transported by oxidation of 10y7 mol of cytochrome C in each reaction tube is about 10 17 electrons. The rate data of Fig. 7 enable us to estimate the electrons normally moved by the reaction, as well as the additional electrons moved by the field. When the control rate is very rapid, about 10 16 electronsrmg proteinrmin, there is no increase due to the field. When the control rate falls to about one third, the 30–40% increase due to the field is about 10 15 electronsrmg proteinrmin ŽIf all the protein were cytochrome oxidase, 1 mg protein would be 3 = 10 15 molecules... Just as found for the Na,K-ATPase, we observe that there is a large positive response to a magnetic field when cytochrome oxidase is not very active, and there is a smaller response at optimal activity. It appears that magnetic fields compete with the normal driving forces, and increase function only when the control level is relatively slow or below normal. This dependence on the biological state also seems to be true in therapeutic applications of magnetic fields w19x. It has always been considered odd that magnetic fields have no apparent effect on normal bones while they can accelerate the healing rate of broken bones. Like the magnetic field effect on the enzyme, therapeutic effects due to magnetic fields appear only when the tissue is damaged and its function diminished. 4.2. The mobile charge interaction (MCI) model
Fig. 9. Variation of the rate constant of the cytochrome oxidase reaction at low magnetic field strengths is shown for the two dialyzed preparations in Fig. 8. Standard errors of the points are in the range of 5–10%.
The mobile charge interaction ŽMCI. model proposes that magnetic fields interact with moving charges, as they would in any conductor, and that the observed dependence on basal enzyme activity is due to field interaction with charge movements during enzyme function. The effects reported here for cytochrome oxidase are similar to earlier results with Na,K-ATPase, and support the MCI interpretation of the data. These studies further show that the relation of signal transduction to enzyme activity applies to
M. Blank, L. Soo r Bioelectrochemistry and Bioenergetics 45 (1998) 253–259 Table 1 Electric and magnetic field thresholds
Na,K-ATPase Cytochrome C oxidase Biosynthesis ŽHL60 cells. Biosynthesis ŽSciara cells. Epidemiological threshold
259
Acknowledgements
Electric field
Magnetic field
0.5 mVrm y - 3 mVrm y
- 0.3 - 0.5 - 0.8 - 0.8 0.2
mT mT mT mT mT
the more complicated case of a non-steady state, equilibrating reaction, where there are simultaneous effects on the oxidation and reduction rate constants. In earlier studies, the qualitative difference between the effects of electric and magnetic fields lent support to the idea of mobile charges. The present studies with cytochrome oxidase also relate activity to the concentration of mobile charges, and show that a field effect is inversely proportional to the number of moving charges. The transient increase in oxidation rate, with no change in the equilibrium, means that oxidation must eventually slow down to normal as reduction speeds up to normal. The effect of the magnetic field is to accelerate the approach to equilibrium. 4.3. Thresholds for changes in cytochrome oxidase function in electric and magnetic fields To characterize biological responses to stimuli, it is customary to establish thresholds and effective ranges experimentally. In magnetic field studies, this process has been inhibited by theoreticians who claim that magnetic field effects are impossible at low field strengths. In view of the low thresholds that have now been found in several independent systems, with better than order of magnitude agreement in the results, this assertion no longer appears tenable. Table 1 shows data from our laboratory of a number of estimated thresholds for changes in enzyme activity stimulated by electric and magnetic fields. There are also data on biosynthesis showing the upper limits of thresholds. The values are consistent, and in the range of thresholds associated with disease in epidemiological studies. We have proposed that interaction of magnetic fields with moving charges could explain stimulation of transcription, where the magnetic field may interact with moving electrons within DNA w20x. Our current results suggest that the magnetic field would enhance charge flow, when the basal flow is relatively small, such as may occur when the cell is in a quiescent state or there is an obstruction Že.g., a kink. in the DNA conducting pathway.
We thank the Heineman Foundation and the NIEHS for support. References w1x M. Blank, ŽEd.., Electromagnetic Fields: Biological Interactions and Mechanisms. Advances in Chemistry, Vol. 250, American Chemical Society Press, Washington, DC, 1995. w2x E.H. Serpersu, T.Y. Tsong, Stimulation of a ouabain-sensitive Rbq uptake in human erythrocytes with an external electric field, J. Membr. Biol. 74 Ž1983. 191–201. w3x E.H. Serpersu, T.Y. Tsong, Activation of electrogenic Rbq transport of ŽNa,K.-ATPase by an electric field, J. Biol. Chem. 259 Ž1984. 7155–7162. w4x M. Blank, L. Soo, The effects of alternating currents on Na,KATPase function, Bioelectrochem. Bioenerg. 22 Ž1989. 313–322. w5x M. Blank, L. Soo, Temperature dependence of electric field effects on the Na,K-ATPase, Bioelectrochem. Bioenerg. 28 Ž1992. 291–299. w6x M. Blank, L. Soo, V. Papstein, Effects of low frequency electromagnetic fields on Na,K-ATPase activity, Bioelectrochem. Bioenerg. 38 Ž1995. 267–273. w7x M. Blank, L. Soo, Frequency dependence of Na,K-ATPase function in magnetic fields, Bioelectrochem. Bioenerg. 42 Ž1997. 231–234. w8x M. Blank, Na,K-ATPase function in alternating electric fields, FASEB J. 6 Ž1992. 2434–2438. w9x M. Blank, Electric and magnetic field signal transduction in the membrane Na,K-ATPase, Adv. Chem. 250 Ž1995. 339–348. w10x M. Blank, L. Soo, The threshold for alternating current inhibition of the Na,K-ATPase, Bioelectromagnetics 13 Ž1992. 329–333. w11x M. Blank, L. Soo, Threshold for Na,K-ATPase stimulation by EM fields, Bioelectrochem. Bioenerg. 40 Ž1996. 63–65. w12x S. Martirosov, M. Blank, Inhibition of F0 F1 -atpase activity in ACfields, Bioelectrochem. Bioenerg. 37 Ž1995. 153–156. w13x D.W. Hilgemann, Channel-like function of the Na,K pump probed at microsecond resolution in giant membrane patches, Science 263 Ž1994. 1429–1431. w14x T. Tsukihara, H. Aoyama, E. Yamashita, T. Tomizaka, H. Yamaguchi, K. Shinzawa-Itoh, R. Nakashima, R. Yaono, S. Yoshikawa, The whole structure of the 13-subunit oxidized cytochrome C oxi˚ Science 272 Ž1996. 1136–1144. dase at 2.8 A, w15x E. Gorczynska, G. Galka, R. Krolikowska, R. Wegrzynowicz, Effect of magnetic field on activity of cytochrome oxidase not moved or moved relative to magnetic field lines, Physiol. Chem. Phys. 14 Ž1982. 201–207. w16x B. Nossol, G. Buse, J. Silny, Influence of weak static and 50 Hz magnetic fields on the redox activity of cytochrome-C oxidase, Bioelectromagnetics 14 Ž1993. 361–372. w17x W.H. Koppenol, C.A.J. Vroonland, R. Braams, The electric potential field around cytochrome C and the effect of ionic strength on reaction rates of horse cytochrome C, Biochim. Biophys. Acta 503 Ž1978. 499–508. w18x L. Smith, Spectrophotometric assay of cytochrome C oxidase, Methods Biochem. Anal. 2 Ž1955. 427–434. w19x C.A.L. Bassett, Bioelectromagnetics in the service of medicine, in: M. Blank, ŽEd.., Electromagnetic Fields: Biological Interactions and Mechanisms, Advances in Chemistry, Vol. 250, 1995, 261–275. w20x M. Blank, R. Goodman, Do electromagnetic fields interact directly with DNA?, Bioelectromagnetics 18 Ž1996. 111–115.