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Electron-electron double resonance (ELDOR) with a loop-gap resonator
JOURNAL
OF MAGNETIC
RESONANCE
63, 142-150 (1985)
Electron-Electron Double Resonance (ELDOR) a Loop-Gap Resonator JAMES S. HYDE, JUN-JIE YIN, W. FRONCISZ,*
AND JIMMY
with
B. FEIX
National Biomedical ESR Center, Department of Radiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53;?26 Received December 18. 1984 Electron-electron double-resonance (ELDOR) experiments on nitroxide-radical-spinlabeled liposomes have been performed using a loop-gap resonator. The signal-to-noise ratio expressed on a molarity basis is 20-fold over the best that has been achieved using a bimodal cavity. This improvement permits ELDOR experiments on spin-labeled plasma membranes of intact cells, as illustrated by a prototype experiment on red blood cells labeled with stearic acid spin label. Moreover, 20 times greater pumping energy density at the sample is achievable for a given incident pump power, permitting ELDOR experiments on less readily saturated systems. Pump and observing frequencies are introduced directly into the loop-gap resonator, which has a relatively low Q, and the pump electron paramagnetic resonance signal is isolated from the receiver using a high Q trap microwave filter. 0 1985 Academic press hc.
Electron-electron double-resonance techniques have not improved very much since the first paper (I). Our interest in ELDOR has increased sharply because of recent studies of translational motions in model membranes (2, 3). However, despite considerable effort, we have not previously been able to use ELDOR to study translational motions in spin-labeled plasma membranes of intact cells-red blood cells in particular. Problems include poor filling factor, unfavorable lipid/water ratio of the cell, microwave heating of the sample, and difficulties in positioning the sample in the bimodal cavity such that dielectric losses were minimized for both pumping and observing resonant modes. These problems have now been overcome using the loop-gap resonator (4) in a novel configuration. The signal-to-noise ratio has been improved by a factor of 20 expressed on a molarity basis compared to that achievable with the bimodal cavity illustrated in Fig. 2 of Ref. (I). (A version of this cavity was used for the work of Refs. (2, 3).) The improvement is a factor of 70 expressed on a “number-of-spins” basis because of the smaller sample volume. This paper describes the apparatus and shows field-swept ELDOR spectra of lipid spin-labeled red blood cells. ELDOR has been reviewed by Kevan and J&pert (5). Two powers must be incident on the sample: a pump power that is sufficient to cause saturation, and an observing power that is generally somewhat below the saturation level. Saturation* On leave from the Department of Biophysics, Institute of Molecular Biology, Jagiellonian University, Krakow, Poland. 0022-2364185 $3.00 Copyright 8 1985 by Academic Press. Inc. All rights of reproduction in any form rcscrvd.
142
ELDOR
WITH
LOOP-GAP
RESONATOR
143
transfer mechanisms cause the observed signal to become partly saturated. It is customary (I, 6) to obtain data at several pump powers and extrapolate to infinite power. The reduction R at infinite pump power, i.e., the fractional change in the observed EPR signal height, is equal in a wide range of circumstances to b = T,,/Tx where T,, is the electron spin-lattice relaxation time and T;’ is the saturation-transfer rate. To determine T,., T,, must be measured independently. In our laboratory the technique of saturation recovery (2, 3, 7, 8) has been used for this purpose. For nitroxide radical spin labels, three mechanisms have heen defined for saturation transfer: nitrogen nuclear relaxation induced by rotational modulation of the electron-nuclear dipolar (END) mechanism, an intramolecular relaxation process (I); Heisenberg exchange, an intermolecular relaxation process (I); and transfer of saturation because of rotational diffusion of spin labels that are nearly immobilized and exhibit rigid limit powder-type spectra, an intramolecular mechanism (9, 10). Heisenberg exchange is of particular interest because exchange rates give direct information on translational diffusion. However, confusion with competing nitrogen nuclear relaxation can arise. Feix et al. (3) introduced the 14N; 15N ELDOR technique. If one species is labeled with one isotope and another species with the other isotope, ELDOR measurements of transfer of saturation between 14N and 15N spectral components directly give Heisenberg exchange information. Nitrogen nuclear relxation cannot couple the two isotopes. Although microwave frequency-swept ELDOR experiments are possible (I 1) field-swept ELDOR is technically much simpler. One sets the two microwave frequencies at the desired separation and sweeps the field. An effect is detected when the pump and observe frequencies are simultaneously on the two resonances between which saturation transfer occurs. Two spectra are obtained: one at zero pump power and one at the desired pump power, permitting immediate determination of R. All work presented here employed the field-swept ELDOR technique. INSTRUMENTATION
The basic idea is simply to feed a normal loop-gap resonator with both pumping and observing microwave frequencies. In the loop-gap resonator, the Q is quite low, of the order of 300, corresponding at X band to 30 MHz between 3 dB points. This low Q is more than compensated by the high energy density for a given input power leading to very good overall performance. Obviously, a compromise is being made-either pump or observing frequencies or both are off the center of the resonant mode. But, as will be shown, overall system performance is so much improved and the intrinsic structure of the resonator so simple compared with a bimodal cavity that this compromise does not seem. overly serious. The microwave circuit is shown in Fig. 1. Pump and observing powers are combined at the magic tee and fed through the circulator to the loop-gap resonator. The pump may be “on resonance” and the observe “off resonance,” or vice versa. Pump power reflected from the resonator carries spurious EPR information and must he separated from observing power that carries the desired information. In the circuit of Fig. 1, the pump power is trapped by the tunable pumptrap cavity. This cavity has a high Q (T&r, mode made from Al, Q0 = 6300) and is strictly matched
144
HYDE
ET AL.
REFERENCE
ARM
TUNABLE PUMP TRAP CAVITY WITH VARIABLE
IRIS
AnENUATOR PHASE SHIFTER DETECTOR CRYSTAL LOAD
FIG. 1. Microwave circuit for ELDER provides the requisite isolation.
with a loop-gap resonator. The tunable pump trap cavity
at the pump frequency, thereby absorbing the pump carrier and the EPR signal sidebands at + 100 kHz on either side of the pump carrier. Observing power is off resonance and reflected from the trap cavity. A total of 50 dB isolation is achievable for a difference frequency of 26 MHz. This corresponds to an EPR signal at the pump frequency that is 300 times less than is observed if the trap is off resonance. This isolation is adequate. The microwave levels incident on the detector crystal are c,r,r~~,,c0s w,t + t,rT,oCOS[(W, f w,)t + &] + C&os(w,t + c/JR) +
w,t
cpi~prT,p~~~ +
+
EprT,pcOs[(Wp
cprd2-4w%t
f
w,)t
+ &,24)
+
6,]
+ qz14~0qwpt
+
+24cosk+
+ 4+) + Wm)t
+
&,2--li3
where subscripts o, p stand for observing and pump frequencies; C,, C, are microwave voltages incident on the circulator; Cn is the reference arm microwave voltages; P and Pr are voltage reflection coefficients of the loop-gap resonator and trap cavity; E at sidebands f w, represents EPR signals; 12-4, Z14 give the voltage isolation between the designated ports of the circulator; phases &, 4n are with respect to w,; and phases &, &,L-4 and &,2-4 with respect to wp. These terms mix in the detector crystal to give rectified outputs at the difference frequency of every possible pair of terms with amplitude proportional to the product of the coefficients of the pair. If Pr,p = 0, then the terms with coefficients $&$C~l-4 and &&Pr,z2-4 provide an experimental limit to the achievable isolation. Tbe l-4 and 2-4 isolations of the waveguide circulator employed here are 50 dB as measured by us on a test bench, exceeding the usual manufacturers’ specification of 40 dB. This isolation happens to be consistent with the measured rejection of the pump EPR signal of 300 times,
ELDOR
WITH LOOP-GAP
RESONATOR
145
indicating that the trapping scheme used here depends not only on the care taken to match the trap cavity, but also and ultimately on the quality of the circulator. It should be possible to improve the isolation further by introduction at arm 4 of another circulator and trap cavity. One sees from the above expression that if PT,r is perfect, then further improvement in isolation is achievable with the pump frequency rather than the observe frequency on resonance, making Pp N 0 and decreasing the term E&&‘JJ~~. Indeed, we have the impression that achievable isolation is somewhat improved when wp is on resonance, although the difference is not great. The term E&&,I,-~ always remains. Since the isolation is not found to depend critically on where the observing frequency is placed on the loop-gap resonance absorption curve, one may generally prefer to have the observe frequency placed on resonance to yield better ELDOR signal intensity. However, if the pumping frequency is too far off resonance, there may be insufficient pump field incident on the sample. In this case, a compromise may be required with observe frequency on one side of resonance and the pump frequency on the other side. An automatic frequency control (AFC) circuit was introduced for the pump power. Its function is to lock the pump frequency to the resonant frequency of the tunable pump-trap cavity. The AFC and trap cavities are of identical construction, and are physically adjacent in order to minimize temperature gradients and improve overall stability. Alternative schemes to lock the pump frequency to the trap cavity itself failed because of conflicting requirements for trapping and for AFC operation. The observe klystron is free running but temperature controlled. The frequency difference as monitored by the counter was stable over extended periods of time to about +2 ppm (+20 kHz). With the observe power off resonance and no observe AFC, one can go back and forth between dispersion and absorption by adjusting the reference arm phase shifter. This is set empirically such that the EPR spectrum corresponds to pure absorption as determined on a normal EPR spectrometer. Figure 2 illustrates the loop-gap resonator and support structure used for these experiments. It is based on Ref. (4). The loop dimensions are 1 mm i.d., 5 mm length. It resonates at 9.1 GHz and has an unloaded Q0 of 460. An optimum aqueous sample has an inner diameter of 0.6 mm (total sample volume 1.4 ~1) and drops the Q about in half. Variable coupling is accomplished by a mechanical arrangement that moves the resonator slightly with respect to the coupling loop which is fixed in space. The entire structure is supported by a fiberglass-epoxy tube 25 cm long, cut away centrally to permit sample access; 100 kHz modulation coils surround the tube but are acoustically isolated. Temperature-controlled gas flows in from the bottom as illustrated and cools the entire structure in the range of 4-40°C. Samples for ELDOR were in TPX (methylpentene polymer; Mitsui Petrochemical Industries, Ltd., Tokyo) capillaries for convenient removal of oxygen (12). PREPARATION
AND SPIN LABELING
OF ERYTHROCYTES
Blood was obtained by venipuncture into heparinized tubes and immediately cooled on ice. Erythrocytes (RBC) were pelleted by centrifugation at 500g for 10
HYDE
146
FIBERGLASS
COUPLING
ET AL.
~
SUPPORT
CLAMP
-
COAXIAL CABLE
II
ADJUST
I I ‘7 SAMPLE ACCESS
SAMPLE SUPPORT HOLDER
THRUST WASHER
REXOLITE HOLDER (FOR SAMPLE AND RESONATOR)
Ag PLATED MACOR COUPLING
-
FIG.
LOOP
EXCHANGE GAS INLET
2. Loop-gap resonator support structure for X band.
min and, after removal of serum, washed three times in phosphate buffered saline (0.145 A4 NaCl, 5 mA4 phosphate, pH 8.0; PBS). Care was taken to remove the buff+ coat after each wash. For spin labeling of erythrocytes, an appropriate volume of stock spin-label solution (e.g., 0.24 mM spin label in chloroform) was placed in a glass vial and the solvent evaporated with a stream of dry nitrogen gas. A 0.2 ml aliquot of washed, intact RBC was added to the vial, and the sample was shaken for 30 min at 37°C. The cells were then centrifuged at 10,OOOg for 1 min and the packed cells used immediately for ELDOR measurements. This procedure gave no hemolysis of red cells and resulted in a negligible amount of free label (see Fig. 4). Spin labels used here were 2-( 14-carboxytetraclecyl)-2-ethyl-4,4-dimethyl-3-oxazolidineoxyl with either 14N or “N at the nitroxide moiety 14NC 16 or 15NC1 6. The 14N derivative was obtained from Syva (Palo Alto, Calif.). The “N derivative was a
ELDOR
WITH LOOP-GAP
RESONATOR
gift from J. H. Park. Dimyristoylphosphalidylcholine were prepared as in Ref. (3).
147
was from Sigma. Liposomes
RESULTS
For r4N ELDOR with labels undergoing fairly fast rotational diffusion, hyperfine lines are 44 MHz apart. The amount of separation could readily be accommodated within the bandpass of the loop-gap resonator. For 14N-r5N field-swept ELDOR, the typical separation is 26 MHz (3), and the loss of observing signal intensity is minimal. Comparison is made in the following paragraphs with the structure of Fig. 2 in Ref. (I), which is a rectangular TELOZmode crossed with a TEloj mode. Figure 3 shows the lines of magnetic flux in this cavity resonator. The magnetic field Ho is at an angle of about 60” with respect to the lines of flux of mode 2. H: (effective) in this mode is four times less than the actual HT. Mode 1 is used as the pump because there is better transition probability and less sample heating for a given pump power. Mode 2 is used for observing, and the factor of 4 loss in sensitivity is very serious. Aqueous samples are difficult to use with this cavity. For samples with high lipid content, typical capillaries are 0.8 mm in diameter. The ratio of incident observe powers comparing the loopgap and bimodal cavity resonators for equal values of H: (effective) at the sample is 50. Typically 80 mW has been used for spin-label studies on liposomal systems with the cavity resonator and 1.5 mW with the loop-gap resonator. These levels represent a compromise between signal intensity and some degree of partial saturation. (As shown in Ref. (I), for small b this compromise makes no difference in R, but as b increases one must be careful about the level of observe power.)
AMIDE
x2
IRIS FOR
FIG. 3. Bimodal cavity used previously for ELDER (Ref. (I)). Lines of magnetic flux are shown (solid line, Mode No. 1; dashed lines, Mode No. 2). Isolation screws, not shown, are used to eliminate spurious coupling between modes. Spurious coupling primarily arises from imperfections in the quartz Dewar insert (also not shown) into which the sample is placed.
148
HYDE
ET
AL.
The ratio of incident pump powers for equal values of H: at the sample is 20. A typical pump power for ELDOR on spin labels with the loop-gap resonator is 50 mW. At 50 mW of pump power, the sample heating is estimated to be +0.3” on a steady-state basis, compared with +O.Y when 400 mW is incident on the bimodal cavity. Noting the factor of 20 difference in energy densities for equal incident powers, it is apparent that sample heating problems are less with the loop-gap resonator than with the bimodal cavity. This may well be due to improved heat transfer from small samples compared with large samples. This steady-state temperature rise depends also on the thermal contact of the sample with the heat-exchange gas that flows over the sample. An advantage of the loop-gap resonator is that one can momentarily raise the pump power to 200 mW corresponding to 10 times greater energy density than is achievable with 400 mW incident power on the bimodal cavity. This permits ELDOR experiments on systems with shorter T,‘s, for example, spin-labeled systems that have not been deoxygenated, such as respiring cells. Experimentally, the ELDOR signal is 20 times better than can be achieved using the bimodal cavity with the comparison done on spin-labeled DMPC liposomal samples of diameters 0.6 mm for the loop-gap resonator and 0.8 mm for the bimodal cavity. This factor is reasonable as can be seen from the following argument: The parameter A is the ratio of peak magnetic fields at constant incident power in two resonant structures (4). Then, if the powers are readjusted such that the fields are the same in the two resonant structures, the EPR signal intensities will vary as A*F’,/V, where V, and V, are the sample volumes in the two structures. The ratio of the sample volumes in the present case is 3.5 where an effective sample length for the bimodal cavity is 1 cm. (The actual length is 2.3 cm but is reduced because of the cosine variation of H, and the modulation amplitude over the sample.) An improvement of a factor of 15 is predicted using the experimental value of 50 for A*, in satisfactory agreement with the experimental factor of 20. Table 1 gives ELDOR results obtained using the loop-gap resonator compared with published results on the same system using the bimodal cavity. The results with the loop-gap resonator are probably somewhat more reliable because of the more uniform observing microwave field over -the sample. The agreement is nevertheless considered good. In this comparison the pump is on resonance. TABLE ELDOR
Reduction
1 Factor,
R-’
DMPC
Bimodal cavity L-G-R ELDOR a Ref. (2). b Ref. (3).
ELDOR
14NC16 (0.5 lu?Jo) 37°C
“NC16.‘5NC16 (0.5;O.i5 A&) 27°C
J%iC~ “NC16.‘5NC16 (0.5;0.;5 Am) 27°C
1.40” 1.40
3.32’ 3.27
3.19 3.14
Blood cells “NC16.‘5NC16 (l;O.5k%) 37°C
3.32 f 0.3
ELDOR
WITH
LOOP-GAP
RESONATOR
149
Reduction at infinite power must theoretically be independent of whether the pump or the observe is on resonance, which has been verified experimentally. Figure 4 shows ELDOR spectra on red blood cells. In the upper trace obtained with a sample containing only 14N spin label, with the doxyl moiety at the 16 position of stearic acid, a very strong intramolecular ELDOR effect is observed that arises from fast nitrogen nuclear relaxation that is induced by motional modulation of the electron-nuclear dipolar interaction. This is a field-swept display with the two frequencies 44 MHz apart corresponding to the separation of the two low-field (M, = + 1, MI = 0) lines. Some motional broadening is evident in the high field M, = -1 spectral fragment and a rather complicated ELDOR response is observed. In the lower trace of Fig. 4, a field-swept ELDOR spectrum is shown using j4N and also “N spin labels with the doxyls at the 16 position and the two frequencies 25 MHz apart. An ELDOR response is observed and the reduction is given in the table. It is substantially smaller than observed in DMPC at the same temperature (37°C) which is above the main phase-transition temperature. CONCLUSION
A solution that appears optimum has been found to the problem of performing ELDOR on spin-labeled biomolecules with good sensitivity and convenience. The main limitation is that sensitivity falls as pump and observing frequencies become increasingly separated. We have considered and built some other loop-gap resonators with potential application to ELDOR including a true crossed or bimodal loop-gap resonator (13) and doubly tuned resonators (14). These should be preferable to the single-mode loop-gap resonator of the present work when the separation of the pump and observing frequencies exceeds about 100 MHz.
a
4 t-lOGI(
AY = 26Mtiz
FIG. 4. ELDOR spectra on red blood cells. The 50% reduction shown in the upper trace arises primarily from nitrogen nuclear relaxation induced by motional modulation of the electron-nuclear dipolar interaction. It is indicative of rotational motions. The 17% reduction shown in the lower trace arises from Heisenkg exchange between 14N and “N moieties and is indicative of translational motion.
150
HYDE
ET
AL.
Development of methodology to permit ELDOR studies on biological membranes has been a principal driving force in the research described here. With some difficulty, we were successful in using the bimodal cavity of Fig. 3 to study intact platelets (work as yet unpublished). These small cells represented the practical limit of applicability of the bimodal cavity. Since the experimental problem primarily is limitation of the amount of plasma membrane, and the membrane-to-cytosol volume ratio varies approximately as (cell radius),-’ improvement of the ELDOR spectrometer signal-to-noise ratio by 20-fold permits the study of cells with 203 greater volume than that of platelets. Thus it seems established that the majority of cell types are now amenable to study with ELDOR. ACKNOWLEDGMEWI-S This work was supported by Grants GM-22923, GM-27665, and RR-01008 from the National Institutes of Health. We thank Professor Jane H. Park of Vanderbilt University for the gift of the 15N spin label. REFERENCES I. J. S. HYDE, J. C. W. CHIEN, AND J. H. FREED, J. Chem. Phys. 48, 42 11 (1968). 2. C. A. POPP AND J. S. HYDE, Pm. Natl. Acad. Sci. USA 79,2559 (1982). 3. J. B. FEIX, C. A. POPP, S. D. VENKATARAMU, A. H. BETH, J. H. PARK, AND J. S. HYDE, Biochemistry 23, 2293 (1984). 4. W. FRONCISZ AND J. S. HYDE, J. Magn. Reson. 47, 5 15 (1982). 5. L. KEVAN AND L. D. KISPERT, “Electron Spin Double Resonance Spectroscopy,” Wiley, New York, 1976. 6. M. P. EASTMAN, G. V. BRUNO, AND J. H. FREED, J. Chern. Phys. 52, 321 (1970). 7. P. W. PERCIVAL AND J. S. HYDE, J. Magn. Reson. 23, 249 (1976). 8. A. KUSUMI, W. K. SUBCZYNSKI, AND J. S. HYDE, Pm. khtl. Acad. Sci. USA 79, 1854 (1982). 9. M. SMIGEL, L. R. DALTON, J. S. HYDE, AND L. A. DALTON, Proc. Natl. Acad. Sci. USA 71, 1925 (1974). 10. J. S. HYDE, M. D. SMIGEL, L. R. DALTON, AND L. A. DALTON, J. Chem. Phys. 62, 1655 (1975). 11. J. S. HYDE, R. C. SNEED, AND G. H. RIST, J. Chem. Phys. 51, 1404 (1969). 12. C. A. POPP AND J. S. HYDE, J. Magn. Reson. 43, 249 (1981). 13. W. FRONCISZ AND J. S. HYDE, U.S. Patent 4,446,429 (1984). 14. M. MEHDUADEH, T. K. ISHII, J. S. HYDE, AND W. FRONCISZ, IEEE Trans. Microwave Theor. Techn. MTT-31, 1059 (1983).
OF MAGNETIC
RESONANCE
63, 142-150 (1985)
Electron-Electron Double Resonance (ELDOR) a Loop-Gap Resonator JAMES S. HYDE, JUN-JIE YIN, W. FRONCISZ,*
AND JIMMY
with
B. FEIX
National Biomedical ESR Center, Department of Radiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53;?26 Received December 18. 1984 Electron-electron double-resonance (ELDOR) experiments on nitroxide-radical-spinlabeled liposomes have been performed using a loop-gap resonator. The signal-to-noise ratio expressed on a molarity basis is 20-fold over the best that has been achieved using a bimodal cavity. This improvement permits ELDOR experiments on spin-labeled plasma membranes of intact cells, as illustrated by a prototype experiment on red blood cells labeled with stearic acid spin label. Moreover, 20 times greater pumping energy density at the sample is achievable for a given incident pump power, permitting ELDOR experiments on less readily saturated systems. Pump and observing frequencies are introduced directly into the loop-gap resonator, which has a relatively low Q, and the pump electron paramagnetic resonance signal is isolated from the receiver using a high Q trap microwave filter. 0 1985 Academic press hc.
Electron-electron double-resonance techniques have not improved very much since the first paper (I). Our interest in ELDOR has increased sharply because of recent studies of translational motions in model membranes (2, 3). However, despite considerable effort, we have not previously been able to use ELDOR to study translational motions in spin-labeled plasma membranes of intact cells-red blood cells in particular. Problems include poor filling factor, unfavorable lipid/water ratio of the cell, microwave heating of the sample, and difficulties in positioning the sample in the bimodal cavity such that dielectric losses were minimized for both pumping and observing resonant modes. These problems have now been overcome using the loop-gap resonator (4) in a novel configuration. The signal-to-noise ratio has been improved by a factor of 20 expressed on a molarity basis compared to that achievable with the bimodal cavity illustrated in Fig. 2 of Ref. (I). (A version of this cavity was used for the work of Refs. (2, 3).) The improvement is a factor of 70 expressed on a “number-of-spins” basis because of the smaller sample volume. This paper describes the apparatus and shows field-swept ELDOR spectra of lipid spin-labeled red blood cells. ELDOR has been reviewed by Kevan and J&pert (5). Two powers must be incident on the sample: a pump power that is sufficient to cause saturation, and an observing power that is generally somewhat below the saturation level. Saturation* On leave from the Department of Biophysics, Institute of Molecular Biology, Jagiellonian University, Krakow, Poland. 0022-2364185 $3.00 Copyright 8 1985 by Academic Press. Inc. All rights of reproduction in any form rcscrvd.
142
ELDOR
WITH
LOOP-GAP
RESONATOR
143
transfer mechanisms cause the observed signal to become partly saturated. It is customary (I, 6) to obtain data at several pump powers and extrapolate to infinite power. The reduction R at infinite pump power, i.e., the fractional change in the observed EPR signal height, is equal in a wide range of circumstances to b = T,,/Tx where T,, is the electron spin-lattice relaxation time and T;’ is the saturation-transfer rate. To determine T,., T,, must be measured independently. In our laboratory the technique of saturation recovery (2, 3, 7, 8) has been used for this purpose. For nitroxide radical spin labels, three mechanisms have heen defined for saturation transfer: nitrogen nuclear relaxation induced by rotational modulation of the electron-nuclear dipolar (END) mechanism, an intramolecular relaxation process (I); Heisenberg exchange, an intermolecular relaxation process (I); and transfer of saturation because of rotational diffusion of spin labels that are nearly immobilized and exhibit rigid limit powder-type spectra, an intramolecular mechanism (9, 10). Heisenberg exchange is of particular interest because exchange rates give direct information on translational diffusion. However, confusion with competing nitrogen nuclear relaxation can arise. Feix et al. (3) introduced the 14N; 15N ELDOR technique. If one species is labeled with one isotope and another species with the other isotope, ELDOR measurements of transfer of saturation between 14N and 15N spectral components directly give Heisenberg exchange information. Nitrogen nuclear relxation cannot couple the two isotopes. Although microwave frequency-swept ELDOR experiments are possible (I 1) field-swept ELDOR is technically much simpler. One sets the two microwave frequencies at the desired separation and sweeps the field. An effect is detected when the pump and observe frequencies are simultaneously on the two resonances between which saturation transfer occurs. Two spectra are obtained: one at zero pump power and one at the desired pump power, permitting immediate determination of R. All work presented here employed the field-swept ELDOR technique. INSTRUMENTATION
The basic idea is simply to feed a normal loop-gap resonator with both pumping and observing microwave frequencies. In the loop-gap resonator, the Q is quite low, of the order of 300, corresponding at X band to 30 MHz between 3 dB points. This low Q is more than compensated by the high energy density for a given input power leading to very good overall performance. Obviously, a compromise is being made-either pump or observing frequencies or both are off the center of the resonant mode. But, as will be shown, overall system performance is so much improved and the intrinsic structure of the resonator so simple compared with a bimodal cavity that this compromise does not seem. overly serious. The microwave circuit is shown in Fig. 1. Pump and observing powers are combined at the magic tee and fed through the circulator to the loop-gap resonator. The pump may be “on resonance” and the observe “off resonance,” or vice versa. Pump power reflected from the resonator carries spurious EPR information and must he separated from observing power that carries the desired information. In the circuit of Fig. 1, the pump power is trapped by the tunable pumptrap cavity. This cavity has a high Q (T&r, mode made from Al, Q0 = 6300) and is strictly matched
144
HYDE
ET AL.
REFERENCE
ARM
TUNABLE PUMP TRAP CAVITY WITH VARIABLE
IRIS
AnENUATOR PHASE SHIFTER DETECTOR CRYSTAL LOAD
FIG. 1. Microwave circuit for ELDER provides the requisite isolation.
with a loop-gap resonator. The tunable pump trap cavity
at the pump frequency, thereby absorbing the pump carrier and the EPR signal sidebands at + 100 kHz on either side of the pump carrier. Observing power is off resonance and reflected from the trap cavity. A total of 50 dB isolation is achievable for a difference frequency of 26 MHz. This corresponds to an EPR signal at the pump frequency that is 300 times less than is observed if the trap is off resonance. This isolation is adequate. The microwave levels incident on the detector crystal are c,r,r~~,,c0s w,t + t,rT,oCOS[(W, f w,)t + &] + C&os(w,t + c/JR) +
w,t
cpi~prT,p~~~ +
+
EprT,pcOs[(Wp
cprd2-4w%t
f
w,)t
+ &,24)
+
6,]
+ qz14~0qwpt
+
+24cosk+
+ 4+) + Wm)t
+
&,2--li3
where subscripts o, p stand for observing and pump frequencies; C,, C, are microwave voltages incident on the circulator; Cn is the reference arm microwave voltages; P and Pr are voltage reflection coefficients of the loop-gap resonator and trap cavity; E at sidebands f w, represents EPR signals; 12-4, Z14 give the voltage isolation between the designated ports of the circulator; phases &, 4n are with respect to w,; and phases &, &,L-4 and &,2-4 with respect to wp. These terms mix in the detector crystal to give rectified outputs at the difference frequency of every possible pair of terms with amplitude proportional to the product of the coefficients of the pair. If Pr,p = 0, then the terms with coefficients $&$C~l-4 and &&Pr,z2-4 provide an experimental limit to the achievable isolation. Tbe l-4 and 2-4 isolations of the waveguide circulator employed here are 50 dB as measured by us on a test bench, exceeding the usual manufacturers’ specification of 40 dB. This isolation happens to be consistent with the measured rejection of the pump EPR signal of 300 times,
ELDOR
WITH LOOP-GAP
RESONATOR
145
indicating that the trapping scheme used here depends not only on the care taken to match the trap cavity, but also and ultimately on the quality of the circulator. It should be possible to improve the isolation further by introduction at arm 4 of another circulator and trap cavity. One sees from the above expression that if PT,r is perfect, then further improvement in isolation is achievable with the pump frequency rather than the observe frequency on resonance, making Pp N 0 and decreasing the term E&&‘JJ~~. Indeed, we have the impression that achievable isolation is somewhat improved when wp is on resonance, although the difference is not great. The term E&&,I,-~ always remains. Since the isolation is not found to depend critically on where the observing frequency is placed on the loop-gap resonance absorption curve, one may generally prefer to have the observe frequency placed on resonance to yield better ELDOR signal intensity. However, if the pumping frequency is too far off resonance, there may be insufficient pump field incident on the sample. In this case, a compromise may be required with observe frequency on one side of resonance and the pump frequency on the other side. An automatic frequency control (AFC) circuit was introduced for the pump power. Its function is to lock the pump frequency to the resonant frequency of the tunable pump-trap cavity. The AFC and trap cavities are of identical construction, and are physically adjacent in order to minimize temperature gradients and improve overall stability. Alternative schemes to lock the pump frequency to the trap cavity itself failed because of conflicting requirements for trapping and for AFC operation. The observe klystron is free running but temperature controlled. The frequency difference as monitored by the counter was stable over extended periods of time to about +2 ppm (+20 kHz). With the observe power off resonance and no observe AFC, one can go back and forth between dispersion and absorption by adjusting the reference arm phase shifter. This is set empirically such that the EPR spectrum corresponds to pure absorption as determined on a normal EPR spectrometer. Figure 2 illustrates the loop-gap resonator and support structure used for these experiments. It is based on Ref. (4). The loop dimensions are 1 mm i.d., 5 mm length. It resonates at 9.1 GHz and has an unloaded Q0 of 460. An optimum aqueous sample has an inner diameter of 0.6 mm (total sample volume 1.4 ~1) and drops the Q about in half. Variable coupling is accomplished by a mechanical arrangement that moves the resonator slightly with respect to the coupling loop which is fixed in space. The entire structure is supported by a fiberglass-epoxy tube 25 cm long, cut away centrally to permit sample access; 100 kHz modulation coils surround the tube but are acoustically isolated. Temperature-controlled gas flows in from the bottom as illustrated and cools the entire structure in the range of 4-40°C. Samples for ELDOR were in TPX (methylpentene polymer; Mitsui Petrochemical Industries, Ltd., Tokyo) capillaries for convenient removal of oxygen (12). PREPARATION
AND SPIN LABELING
OF ERYTHROCYTES
Blood was obtained by venipuncture into heparinized tubes and immediately cooled on ice. Erythrocytes (RBC) were pelleted by centrifugation at 500g for 10
HYDE
146
FIBERGLASS
COUPLING
ET AL.
~
SUPPORT
CLAMP
-
COAXIAL CABLE
II
ADJUST
I I ‘7 SAMPLE ACCESS
SAMPLE SUPPORT HOLDER
THRUST WASHER
REXOLITE HOLDER (FOR SAMPLE AND RESONATOR)
Ag PLATED MACOR COUPLING
-
FIG.
LOOP
EXCHANGE GAS INLET
2. Loop-gap resonator support structure for X band.
min and, after removal of serum, washed three times in phosphate buffered saline (0.145 A4 NaCl, 5 mA4 phosphate, pH 8.0; PBS). Care was taken to remove the buff+ coat after each wash. For spin labeling of erythrocytes, an appropriate volume of stock spin-label solution (e.g., 0.24 mM spin label in chloroform) was placed in a glass vial and the solvent evaporated with a stream of dry nitrogen gas. A 0.2 ml aliquot of washed, intact RBC was added to the vial, and the sample was shaken for 30 min at 37°C. The cells were then centrifuged at 10,OOOg for 1 min and the packed cells used immediately for ELDOR measurements. This procedure gave no hemolysis of red cells and resulted in a negligible amount of free label (see Fig. 4). Spin labels used here were 2-( 14-carboxytetraclecyl)-2-ethyl-4,4-dimethyl-3-oxazolidineoxyl with either 14N or “N at the nitroxide moiety 14NC 16 or 15NC1 6. The 14N derivative was obtained from Syva (Palo Alto, Calif.). The “N derivative was a
ELDOR
WITH LOOP-GAP
RESONATOR
gift from J. H. Park. Dimyristoylphosphalidylcholine were prepared as in Ref. (3).
147
was from Sigma. Liposomes
RESULTS
For r4N ELDOR with labels undergoing fairly fast rotational diffusion, hyperfine lines are 44 MHz apart. The amount of separation could readily be accommodated within the bandpass of the loop-gap resonator. For 14N-r5N field-swept ELDOR, the typical separation is 26 MHz (3), and the loss of observing signal intensity is minimal. Comparison is made in the following paragraphs with the structure of Fig. 2 in Ref. (I), which is a rectangular TELOZmode crossed with a TEloj mode. Figure 3 shows the lines of magnetic flux in this cavity resonator. The magnetic field Ho is at an angle of about 60” with respect to the lines of flux of mode 2. H: (effective) in this mode is four times less than the actual HT. Mode 1 is used as the pump because there is better transition probability and less sample heating for a given pump power. Mode 2 is used for observing, and the factor of 4 loss in sensitivity is very serious. Aqueous samples are difficult to use with this cavity. For samples with high lipid content, typical capillaries are 0.8 mm in diameter. The ratio of incident observe powers comparing the loopgap and bimodal cavity resonators for equal values of H: (effective) at the sample is 50. Typically 80 mW has been used for spin-label studies on liposomal systems with the cavity resonator and 1.5 mW with the loop-gap resonator. These levels represent a compromise between signal intensity and some degree of partial saturation. (As shown in Ref. (I), for small b this compromise makes no difference in R, but as b increases one must be careful about the level of observe power.)
AMIDE
x2
IRIS FOR
FIG. 3. Bimodal cavity used previously for ELDER (Ref. (I)). Lines of magnetic flux are shown (solid line, Mode No. 1; dashed lines, Mode No. 2). Isolation screws, not shown, are used to eliminate spurious coupling between modes. Spurious coupling primarily arises from imperfections in the quartz Dewar insert (also not shown) into which the sample is placed.
148
HYDE
ET
AL.
The ratio of incident pump powers for equal values of H: at the sample is 20. A typical pump power for ELDOR on spin labels with the loop-gap resonator is 50 mW. At 50 mW of pump power, the sample heating is estimated to be +0.3” on a steady-state basis, compared with +O.Y when 400 mW is incident on the bimodal cavity. Noting the factor of 20 difference in energy densities for equal incident powers, it is apparent that sample heating problems are less with the loop-gap resonator than with the bimodal cavity. This may well be due to improved heat transfer from small samples compared with large samples. This steady-state temperature rise depends also on the thermal contact of the sample with the heat-exchange gas that flows over the sample. An advantage of the loop-gap resonator is that one can momentarily raise the pump power to 200 mW corresponding to 10 times greater energy density than is achievable with 400 mW incident power on the bimodal cavity. This permits ELDOR experiments on systems with shorter T,‘s, for example, spin-labeled systems that have not been deoxygenated, such as respiring cells. Experimentally, the ELDOR signal is 20 times better than can be achieved using the bimodal cavity with the comparison done on spin-labeled DMPC liposomal samples of diameters 0.6 mm for the loop-gap resonator and 0.8 mm for the bimodal cavity. This factor is reasonable as can be seen from the following argument: The parameter A is the ratio of peak magnetic fields at constant incident power in two resonant structures (4). Then, if the powers are readjusted such that the fields are the same in the two resonant structures, the EPR signal intensities will vary as A*F’,/V, where V, and V, are the sample volumes in the two structures. The ratio of the sample volumes in the present case is 3.5 where an effective sample length for the bimodal cavity is 1 cm. (The actual length is 2.3 cm but is reduced because of the cosine variation of H, and the modulation amplitude over the sample.) An improvement of a factor of 15 is predicted using the experimental value of 50 for A*, in satisfactory agreement with the experimental factor of 20. Table 1 gives ELDOR results obtained using the loop-gap resonator compared with published results on the same system using the bimodal cavity. The results with the loop-gap resonator are probably somewhat more reliable because of the more uniform observing microwave field over -the sample. The agreement is nevertheless considered good. In this comparison the pump is on resonance. TABLE ELDOR
Reduction
1 Factor,
R-’
DMPC
Bimodal cavity L-G-R ELDOR a Ref. (2). b Ref. (3).
ELDOR
14NC16 (0.5 lu?Jo) 37°C
“NC16.‘5NC16 (0.5;O.i5 A&) 27°C
J%iC~ “NC16.‘5NC16 (0.5;0.;5 Am) 27°C
1.40” 1.40
3.32’ 3.27
3.19 3.14
Blood cells “NC16.‘5NC16 (l;O.5k%) 37°C
3.32 f 0.3
ELDOR
WITH
LOOP-GAP
RESONATOR
149
Reduction at infinite power must theoretically be independent of whether the pump or the observe is on resonance, which has been verified experimentally. Figure 4 shows ELDOR spectra on red blood cells. In the upper trace obtained with a sample containing only 14N spin label, with the doxyl moiety at the 16 position of stearic acid, a very strong intramolecular ELDOR effect is observed that arises from fast nitrogen nuclear relaxation that is induced by motional modulation of the electron-nuclear dipolar interaction. This is a field-swept display with the two frequencies 44 MHz apart corresponding to the separation of the two low-field (M, = + 1, MI = 0) lines. Some motional broadening is evident in the high field M, = -1 spectral fragment and a rather complicated ELDOR response is observed. In the lower trace of Fig. 4, a field-swept ELDOR spectrum is shown using j4N and also “N spin labels with the doxyls at the 16 position and the two frequencies 25 MHz apart. An ELDOR response is observed and the reduction is given in the table. It is substantially smaller than observed in DMPC at the same temperature (37°C) which is above the main phase-transition temperature. CONCLUSION
A solution that appears optimum has been found to the problem of performing ELDOR on spin-labeled biomolecules with good sensitivity and convenience. The main limitation is that sensitivity falls as pump and observing frequencies become increasingly separated. We have considered and built some other loop-gap resonators with potential application to ELDOR including a true crossed or bimodal loop-gap resonator (13) and doubly tuned resonators (14). These should be preferable to the single-mode loop-gap resonator of the present work when the separation of the pump and observing frequencies exceeds about 100 MHz.
a
4 t-lOGI(
AY = 26Mtiz
FIG. 4. ELDOR spectra on red blood cells. The 50% reduction shown in the upper trace arises primarily from nitrogen nuclear relaxation induced by motional modulation of the electron-nuclear dipolar interaction. It is indicative of rotational motions. The 17% reduction shown in the lower trace arises from Heisenkg exchange between 14N and “N moieties and is indicative of translational motion.
150
HYDE
ET
AL.
Development of methodology to permit ELDOR studies on biological membranes has been a principal driving force in the research described here. With some difficulty, we were successful in using the bimodal cavity of Fig. 3 to study intact platelets (work as yet unpublished). These small cells represented the practical limit of applicability of the bimodal cavity. Since the experimental problem primarily is limitation of the amount of plasma membrane, and the membrane-to-cytosol volume ratio varies approximately as (cell radius),-’ improvement of the ELDOR spectrometer signal-to-noise ratio by 20-fold permits the study of cells with 203 greater volume than that of platelets. Thus it seems established that the majority of cell types are now amenable to study with ELDOR. ACKNOWLEDGMEWI-S This work was supported by Grants GM-22923, GM-27665, and RR-01008 from the National Institutes of Health. We thank Professor Jane H. Park of Vanderbilt University for the gift of the 15N spin label. REFERENCES I. J. S. HYDE, J. C. W. CHIEN, AND J. H. FREED, J. Chem. Phys. 48, 42 11 (1968). 2. C. A. POPP AND J. S. HYDE, Pm. Natl. Acad. Sci. USA 79,2559 (1982). 3. J. B. FEIX, C. A. POPP, S. D. VENKATARAMU, A. H. BETH, J. H. PARK, AND J. S. HYDE, Biochemistry 23, 2293 (1984). 4. W. FRONCISZ AND J. S. HYDE, J. Magn. Reson. 47, 5 15 (1982). 5. L. KEVAN AND L. D. KISPERT, “Electron Spin Double Resonance Spectroscopy,” Wiley, New York, 1976. 6. M. P. EASTMAN, G. V. BRUNO, AND J. H. FREED, J. Chern. Phys. 52, 321 (1970). 7. P. W. PERCIVAL AND J. S. HYDE, J. Magn. Reson. 23, 249 (1976). 8. A. KUSUMI, W. K. SUBCZYNSKI, AND J. S. HYDE, Pm. khtl. Acad. Sci. USA 79, 1854 (1982). 9. M. SMIGEL, L. R. DALTON, J. S. HYDE, AND L. A. DALTON, Proc. Natl. Acad. Sci. USA 71, 1925 (1974). 10. J. S. HYDE, M. D. SMIGEL, L. R. DALTON, AND L. A. DALTON, J. Chem. Phys. 62, 1655 (1975). 11. J. S. HYDE, R. C. SNEED, AND G. H. RIST, J. Chem. Phys. 51, 1404 (1969). 12. C. A. POPP AND J. S. HYDE, J. Magn. Reson. 43, 249 (1981). 13. W. FRONCISZ AND J. S. HYDE, U.S. Patent 4,446,429 (1984). 14. M. MEHDUADEH, T. K. ISHII, J. S. HYDE, AND W. FRONCISZ, IEEE Trans. Microwave Theor. Techn. MTT-31, 1059 (1983).