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Dynamic range problem in Fourier transform NMR. Modified WEFT pulse sequence
JOURNAL
OF MAGNETIC
RESONANCE
24,461-465
(1976)
Dynamic RangeProblem in Fourier Transform NMR. Modified WEFT PulseSequence* The dynamic range problem encountered in the use of Fourier transform NMR in aqueous solutions is well documented (I-12). Since water is the solvent chosen by nature for biochemical reactions in vivo, most in vitro studies aimed at understanding the structure and mechanism of action of biochemical molecules must also be carried out in aqueous solvents. Deuteration leads to a loss of the resonances of exchangeable protons (13) and does leave a residual water signal which often results in dynamic range problems. The dynamic range problem in working with H,O solutions was first considered by Redfield and the author (I, 2) and a solution to this problem based on the use of long selective pulses to resonate the spins of interest without exciting the water resonance was developed by these authors (I, 2). Several other methods of handling the dynamic range problem have recently emerged in the literature (3-22) with varying degree of success. One of these, the “Water Eliminated Fourier Transform” (WEFT) technique, originally suggested by Patt and Sykes (3), is based on the differences between the spinlattice relaxation times of the protons of interest and those of H,O or HDO (3-6). A major advantage of the WEFT sequence for recording spectra in H,O or HDO solutions is that it is easily adapta.ble to modern commercial FT-NMR spectrometers. Several disadvantages of this technique have, however, become obvious : (1) The relative intensities of the resonances may be significantly distorted by the use of WEFT. Its use therefore precludes quantitative studies based on the area under resonance absorptions. (2) Since in WEFT, one is essentially recording partially relaxed spectra as opposed to fully relaxed ones, the overall sensitivity of the method is suboptimal, an undesirable feature for much biochemical work where sample quantities are limited and optimum S/N ratio is an important consideration. (3) The intensities of exchangeable protons observed by this technique will be further distorted by the presence of crossrelaxation and chemical-exchange effects. (4) The technique is not easily amenable to time-resolved studies such as measurements of spin-lattice relaxation times. Signal elimination by solvent saturation, another technique for overcoming the dynamic range problem, has also heen in use recently (7,8, 24); its principal disadvantages are that exchangeable protons will have their intensities distorted by cross relaxation or chemical exchange, and that the extent of signal suppression is often not sufficient to observe samples in the millimolar range with optimum S/N ratio. The only other technique widely in use for overcoming the dynamic range problems is rapid scan correlation spectroscopy (9, 10). This technique has recently been extended to carry out r, measurements (15), but its usefulness in such studies with complex spectra appears at * This workwassupported by Nationalhstitutesof HealthGrants lROl-AM-HL-19454, AM-13351, by Grants to the Institute for CancerResearch,CA-06927, RR-05539, from the National Institutes of Health, and by an appropriation from the Commonwealth of Pennsylvania. Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
461
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COMMUNICATIONS
present more limited. Although long pulse techniques (1,2, II) may become the method of choice for future studies in water, their use is currently limited, to the knowledge of this author, only to one laboratory (II). The purpose of this communication is to report a modified WEFT technique which is essentially free from the major disadvantages of the conventional WEFT (3-6) technique but which retains the ease of adaptability on most modern NMR spectrometers and requires no hardware modifications. The technique, unlike regular WEFT, should be useful in routine spin-lattice relaxation studies in aqueous solutions as well. The pulse sequence for modified WEFT is shown below:
All spins are allowed to equilibrate for a recovery time, PD (-5T,). A long weak nulling pulse of duration Tin resonance with water spins (at vHZO)is then applied to invert the water magnetization in the rotating frame by -180”, followed by a waiting time z,,,i to allow the longitudinal component of water magnetization to reach its null. At the null point of H,O, a short 90” pulse samples the magnetization of all spins over the spectral region of interest. The free induction signal acquired during the acquisition time (AT) is Fourier transformed in the usual manner. A homogeneity-spoiling (HS) pulse lasting for
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463
water magnetization and the null point of the water signal was determined empirically. The sample under examination was 0.1% ethanol in 95 % Hz0 + 5 % D,O (to allow field-frequency locking on deuterium) doped with 0.1 mM MnCl, to shorten relaxation times. The spectrum in Fig. 1 wa;s obtained in 200 sec. The S/N measured on the methy-
1
FIG. 1. NMR spectrum of 0.1% ethanol in 95 % HZ0 + 5 % D,O doped with 0.1 mA4 MnClz after time averaging 25 transients of free induction signal in a total time of 200 set using modified WEFT technique. lene quartet is comparable to that obtained in Ref. (I) using the long pulse techniques
in the same total time. The residual H,O signal after a rough determination of the null point in our system corresponded to -300 mM protons. With more careful determination of the null point for HzO, better results should be possible. The noise in the spectrum is entirely thermal and the level of nulling achieved is quite sufficient for carrying out studies on solutions in the range of -1 mM. In Fig. 2, we show a comparison of the
FIG. 2. Comparison of single transient spectra obtained using modified WEFT (bottom trace) and conventional WEFT (upper trace) te~zhniques. Sample conditions were the same as in Fig. 1. The small broad line at the left and of the lower trace is a spinning side band of H20.
464
COMMUNICATIONS
performance of modified WEFT and regular WEFT in a single transient experiment. As is quite obvious, the use of regular WEFT on the sample under examination results in poor signal-to-noise and visually distorted relative intensities. On the other hand, modified WEFT reproduces the ethanol spectrum quite faithfully. Although named here as modified WEFT, the basis of Hz0 resonance elimination in the present technique is quite different from that in WEFT. For example, the present technique is not dependent on the differential relaxation behavior of H,O and the protons of interest and hence is applicable to systems with any T1 value for water, particularly in working with paramagnetic biochemical systems where water Tl may be considerably shortened by electron-nuclear interactions (14). Further, since the choice of nulling pulse length is limited only by the separation of the resonances of interest from water, the only requirement being that the nulling pulse not affect the spins of interest, it could be made arbitrarily short (~10 msec) for observing exchangeable proton resonances well downfield from H,O (> 100 Hz). Thus it should be quite useful for observing tryptophan NH, imidazole NH, and tyrosine OH protons. Since r,,ii could be arbitrarily shortened to any value (220 msec) by choosing the nulling pulse flip angle appropriately, it is possible to eliminate intensity distortions due to cross-relaxation and saturationtransfer effects which occur on a time scale of 7 100 msec. Interestingly, in favorable cases it may be possible to determine proton-exchange or cross-relaxation rates by studying the intensities of exchangeable proton resonances as a function of ~,,~rr, adjusting the nulling nutation angle in the range 90-180” appropriately according to Eq. [I]. The modified WEFT technique should be easily adaptable to spin-lattice relaxation measurements with minor software modificati0ns.l Such studies are in progress. ACKNOWLEDGMENTS The author thanks Professors A. S. Mildvan, A. G. Redfield, E. D. Becker, and Dr. B. L. Bean for their helpful comments. REFERENCES 1. A. G. REDFIELD AND R. K. GUPTA, J. Chem. Phys. 54,1418 (1971). 2. A. G. REDFIELD AND R. K. GUPTA, Advun. Mugn. Resonance 5,81 (1971). 3. S. L. PATT AND B. D. SYKES, J. Chem. Phys. 56,3182 (1972). 4. F. W. BENZ, J. FEENEY, AND G. C. K. ROBERTS, J. iI4agn. Resonance 8,114 (1972). 5. E. S. MOOBERRY AND T. R. KRUGH, J. Magn. Resonance 17,128 (1975). 6. T. R. KRUGH AND W. C. SCHAEFER, J. Mugn. Resonance 19,99 (1975). 7. J. P. JESSON, P. MEAKIN, AND G. KNEISSEL, J. Amer. Chem. Sot. 95, 618 (1973). 8. H. E. BLEICH AND J. A. GLASEL, J. Magn. Resonance 18,401 (1975). 9. J. DADOK AND R. F. SPRECHER, J. Magn. Resonance 13,243 (1974).
1 Two long and two short rf pulses are needed for an inversion-recovery T1 measurement. After equilibration the water spins are subjected to a long 180” pulse immediately followed by a short 180” pulse which returns water magnetization to the longitudinal direction in the rotating frame, the long and short pulses together making water execute a 2n nutation. The proton spins of interest are unaffected by the long pulse but are inverted by the short 180” pulse. A second long pulse of nutation angle 0 at the H,O frequency followed by homogeneity spoiling nulls the water signal a time rntnu,,later according to Eq. [l]. A short 90” pulse then samples the partially relaxed state of spins under observation at the null point of the water signal. The spins of interest undergo the standard inversion-recovery sequence, being unaffected by the long pulses. The time interval between the short 180” and 90” pulses is varied to obtain partially relaxed spectra and TI behavior of the spins of interest in the usual manner.
COMMUNICATIONS
465
J. Magn. Resonance 13,275 (1974). J. Magn. Resonance 19,114 (1975). 12. W. HI.LL, J. Chem. P&s. 59,1775 (1973). 13. R. K. GUPTA AND J. M. PESANDO,,I. Biol. Chem. 250,263O (1975). 14. R. K. GUPTA, C. H. FUNG, AND A. S. MILDVAN, J. Biol. Chem. 251,242l (1976). 15. R. K. GUPTA, J. A. FERRETTI, AND E. D. BECKER, J. Magn. Resonance 16,505 (1974). 10. II.
R. K. GUPTA, J. A. FERRETTI, A. G. REDFIELD, S. D. KUNZ, B. L. TOMLINSON AND H. D.
AND E. D. BECKER, AND E. K. RALPH,
RAJ K. GUPTA~ The Institute,for Cancer Research Fox Chase Cancer Center Philadelphia, Pennsylvania 191 I I Received September 7, 1976 i Research Career Development Awardee l-K04-AM-HL00231
17
of the National Institutes of Health.
OF MAGNETIC
RESONANCE
24,461-465
(1976)
Dynamic RangeProblem in Fourier Transform NMR. Modified WEFT PulseSequence* The dynamic range problem encountered in the use of Fourier transform NMR in aqueous solutions is well documented (I-12). Since water is the solvent chosen by nature for biochemical reactions in vivo, most in vitro studies aimed at understanding the structure and mechanism of action of biochemical molecules must also be carried out in aqueous solvents. Deuteration leads to a loss of the resonances of exchangeable protons (13) and does leave a residual water signal which often results in dynamic range problems. The dynamic range problem in working with H,O solutions was first considered by Redfield and the author (I, 2) and a solution to this problem based on the use of long selective pulses to resonate the spins of interest without exciting the water resonance was developed by these authors (I, 2). Several other methods of handling the dynamic range problem have recently emerged in the literature (3-22) with varying degree of success. One of these, the “Water Eliminated Fourier Transform” (WEFT) technique, originally suggested by Patt and Sykes (3), is based on the differences between the spinlattice relaxation times of the protons of interest and those of H,O or HDO (3-6). A major advantage of the WEFT sequence for recording spectra in H,O or HDO solutions is that it is easily adapta.ble to modern commercial FT-NMR spectrometers. Several disadvantages of this technique have, however, become obvious : (1) The relative intensities of the resonances may be significantly distorted by the use of WEFT. Its use therefore precludes quantitative studies based on the area under resonance absorptions. (2) Since in WEFT, one is essentially recording partially relaxed spectra as opposed to fully relaxed ones, the overall sensitivity of the method is suboptimal, an undesirable feature for much biochemical work where sample quantities are limited and optimum S/N ratio is an important consideration. (3) The intensities of exchangeable protons observed by this technique will be further distorted by the presence of crossrelaxation and chemical-exchange effects. (4) The technique is not easily amenable to time-resolved studies such as measurements of spin-lattice relaxation times. Signal elimination by solvent saturation, another technique for overcoming the dynamic range problem, has also heen in use recently (7,8, 24); its principal disadvantages are that exchangeable protons will have their intensities distorted by cross relaxation or chemical exchange, and that the extent of signal suppression is often not sufficient to observe samples in the millimolar range with optimum S/N ratio. The only other technique widely in use for overcoming the dynamic range problems is rapid scan correlation spectroscopy (9, 10). This technique has recently been extended to carry out r, measurements (15), but its usefulness in such studies with complex spectra appears at * This workwassupported by Nationalhstitutesof HealthGrants lROl-AM-HL-19454, AM-13351, by Grants to the Institute for CancerResearch,CA-06927, RR-05539, from the National Institutes of Health, and by an appropriation from the Commonwealth of Pennsylvania. Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
461
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COMMUNICATIONS
present more limited. Although long pulse techniques (1,2, II) may become the method of choice for future studies in water, their use is currently limited, to the knowledge of this author, only to one laboratory (II). The purpose of this communication is to report a modified WEFT technique which is essentially free from the major disadvantages of the conventional WEFT (3-6) technique but which retains the ease of adaptability on most modern NMR spectrometers and requires no hardware modifications. The technique, unlike regular WEFT, should be useful in routine spin-lattice relaxation studies in aqueous solutions as well. The pulse sequence for modified WEFT is shown below:
All spins are allowed to equilibrate for a recovery time, PD (-5T,). A long weak nulling pulse of duration Tin resonance with water spins (at vHZO)is then applied to invert the water magnetization in the rotating frame by -180”, followed by a waiting time z,,,i to allow the longitudinal component of water magnetization to reach its null. At the null point of H,O, a short 90” pulse samples the magnetization of all spins over the spectral region of interest. The free induction signal acquired during the acquisition time (AT) is Fourier transformed in the usual manner. A homogeneity-spoiling (HS) pulse lasting for
COMMUNICATIONS
463
water magnetization and the null point of the water signal was determined empirically. The sample under examination was 0.1% ethanol in 95 % Hz0 + 5 % D,O (to allow field-frequency locking on deuterium) doped with 0.1 mM MnCl, to shorten relaxation times. The spectrum in Fig. 1 wa;s obtained in 200 sec. The S/N measured on the methy-
1
FIG. 1. NMR spectrum of 0.1% ethanol in 95 % HZ0 + 5 % D,O doped with 0.1 mA4 MnClz after time averaging 25 transients of free induction signal in a total time of 200 set using modified WEFT technique. lene quartet is comparable to that obtained in Ref. (I) using the long pulse techniques
in the same total time. The residual H,O signal after a rough determination of the null point in our system corresponded to -300 mM protons. With more careful determination of the null point for HzO, better results should be possible. The noise in the spectrum is entirely thermal and the level of nulling achieved is quite sufficient for carrying out studies on solutions in the range of -1 mM. In Fig. 2, we show a comparison of the
FIG. 2. Comparison of single transient spectra obtained using modified WEFT (bottom trace) and conventional WEFT (upper trace) te~zhniques. Sample conditions were the same as in Fig. 1. The small broad line at the left and of the lower trace is a spinning side band of H20.
464
COMMUNICATIONS
performance of modified WEFT and regular WEFT in a single transient experiment. As is quite obvious, the use of regular WEFT on the sample under examination results in poor signal-to-noise and visually distorted relative intensities. On the other hand, modified WEFT reproduces the ethanol spectrum quite faithfully. Although named here as modified WEFT, the basis of Hz0 resonance elimination in the present technique is quite different from that in WEFT. For example, the present technique is not dependent on the differential relaxation behavior of H,O and the protons of interest and hence is applicable to systems with any T1 value for water, particularly in working with paramagnetic biochemical systems where water Tl may be considerably shortened by electron-nuclear interactions (14). Further, since the choice of nulling pulse length is limited only by the separation of the resonances of interest from water, the only requirement being that the nulling pulse not affect the spins of interest, it could be made arbitrarily short (~10 msec) for observing exchangeable proton resonances well downfield from H,O (> 100 Hz). Thus it should be quite useful for observing tryptophan NH, imidazole NH, and tyrosine OH protons. Since r,,ii could be arbitrarily shortened to any value (220 msec) by choosing the nulling pulse flip angle appropriately, it is possible to eliminate intensity distortions due to cross-relaxation and saturationtransfer effects which occur on a time scale of 7 100 msec. Interestingly, in favorable cases it may be possible to determine proton-exchange or cross-relaxation rates by studying the intensities of exchangeable proton resonances as a function of ~,,~rr, adjusting the nulling nutation angle in the range 90-180” appropriately according to Eq. [I]. The modified WEFT technique should be easily adaptable to spin-lattice relaxation measurements with minor software modificati0ns.l Such studies are in progress. ACKNOWLEDGMENTS The author thanks Professors A. S. Mildvan, A. G. Redfield, E. D. Becker, and Dr. B. L. Bean for their helpful comments. REFERENCES 1. A. G. REDFIELD AND R. K. GUPTA, J. Chem. Phys. 54,1418 (1971). 2. A. G. REDFIELD AND R. K. GUPTA, Advun. Mugn. Resonance 5,81 (1971). 3. S. L. PATT AND B. D. SYKES, J. Chem. Phys. 56,3182 (1972). 4. F. W. BENZ, J. FEENEY, AND G. C. K. ROBERTS, J. iI4agn. Resonance 8,114 (1972). 5. E. S. MOOBERRY AND T. R. KRUGH, J. Magn. Resonance 17,128 (1975). 6. T. R. KRUGH AND W. C. SCHAEFER, J. Mugn. Resonance 19,99 (1975). 7. J. P. JESSON, P. MEAKIN, AND G. KNEISSEL, J. Amer. Chem. Sot. 95, 618 (1973). 8. H. E. BLEICH AND J. A. GLASEL, J. Magn. Resonance 18,401 (1975). 9. J. DADOK AND R. F. SPRECHER, J. Magn. Resonance 13,243 (1974).
1 Two long and two short rf pulses are needed for an inversion-recovery T1 measurement. After equilibration the water spins are subjected to a long 180” pulse immediately followed by a short 180” pulse which returns water magnetization to the longitudinal direction in the rotating frame, the long and short pulses together making water execute a 2n nutation. The proton spins of interest are unaffected by the long pulse but are inverted by the short 180” pulse. A second long pulse of nutation angle 0 at the H,O frequency followed by homogeneity spoiling nulls the water signal a time rntnu,,later according to Eq. [l]. A short 90” pulse then samples the partially relaxed state of spins under observation at the null point of the water signal. The spins of interest undergo the standard inversion-recovery sequence, being unaffected by the long pulses. The time interval between the short 180” and 90” pulses is varied to obtain partially relaxed spectra and TI behavior of the spins of interest in the usual manner.
COMMUNICATIONS
465
J. Magn. Resonance 13,275 (1974). J. Magn. Resonance 19,114 (1975). 12. W. HI.LL, J. Chem. P&s. 59,1775 (1973). 13. R. K. GUPTA AND J. M. PESANDO,,I. Biol. Chem. 250,263O (1975). 14. R. K. GUPTA, C. H. FUNG, AND A. S. MILDVAN, J. Biol. Chem. 251,242l (1976). 15. R. K. GUPTA, J. A. FERRETTI, AND E. D. BECKER, J. Magn. Resonance 16,505 (1974). 10. II.
R. K. GUPTA, J. A. FERRETTI, A. G. REDFIELD, S. D. KUNZ, B. L. TOMLINSON AND H. D.
AND E. D. BECKER, AND E. K. RALPH,
RAJ K. GUPTA~ The Institute,for Cancer Research Fox Chase Cancer Center Philadelphia, Pennsylvania 191 I I Received September 7, 1976 i Research Career Development Awardee l-K04-AM-HL00231
17
of the National Institutes of Health.