- Email: [email protected]
A magnesium(II)ATP thermometer for 31P NMR studies of biological systems
JOURNAL
OF MAGNETIC
RESONANCE
40, 587-589
(1980)
A Magnesium(II)ATP Thermometer for 31PNMR Studies of Biological Systems * A common problem in 31P NMR studies of biological systems is accurate measurement of sample temperature. Reliable temperature measurements require either a thermocouple placed in the sample itself (or in a sample of similar geometry and dielectric constant) or the use of a suitable compound having a temperature-dependent chemical shift. Difficulties in placing a thermocouple in the NMR sample tube, especially with superconducting spectrometers, and the lack of suitable compounds having temperature-dependent shielding characteristics for every nucleus studied have resulted in the use of a thermocouple placed either just below or above the receiver/transmitter coil for the measurement of flow gas temperature which may not be the same as the sample temperature. Such temperature readings become especially unreliable when high-power decoupling is used, which is usually the case with 31P NMR studies. There is also some evidence of errors in thermocouple measurements resulting from superconducting magnetic fields (1). Several lH, 2H, lgF, 13C, and 5gCo thermometers have been proposed previously (2-7). To use these existing nucleus-specific thermometers in 31P NMR studies, one needs to switch back and forth between the 31P nucleus and the thermometer nucleus for temperature measurements. This operation generally requires probe retuning and is inconvenient for variable-temperature operation even with broadband design NMR probes. Because of the widespread use of 31P NMR in biological research, it is desirable to have an NMR thermometer based on the 31P nucleus for convenient measurement of sample temperature in biological applications of 31P NMR. In this communication we propose a MgATP thermometer for 31P N studies. In the course of our investigations on the state of ionized magnesium in intact cells by the noninvasive 31P NMR spectroscopy (8-IO), we have found that the chemical shift difference between the aP and pP resonances in neutral pH solutions of MgATP (6f#ATP), which is independent of ionic strength and small variations in pH, shows a sizable temperature dependence. Since 31P resonances of ATP are sharp and narrow (51 Hz), and ATP is very soluble in aqueous medium to permit the attainment of a good signal-to-noise ratio in a few minutes of time averaging, it is possible to measure the chemical shift difference @$ATP with an accuracy of a few tenths of a hertz. Our own measurements of this difference on a given MgATP sample at 40.5 MHz (on a Varian XL-100) using 8000 data points and a spectral width of 1000 Hz are reproducible to 5 kO.2 Hz. * This work has been supported by NIH Grants AM-19454 RR-05539 to the Institute for Cancer Research from the appropriation from the Commonwealth of Pennsylvania.
and AM-13351, National Institutes
by Grants CA-06927 and of Health, and by an
587 0022-2364/X0/120587-03$02.00!0 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
588
COMMUNICATIONS
5
IO I5 Chemical Shift (ppm)
io
FIG. 1. A 31P NMR spectrum showing the aP, /3P, and yP resonances of MgATP at 40.5 MHz (pH 7.2, p = 0.15 M, and T = 37°C). The sample contained 10 mM ATP and 30 m/M MgC12. The quantity 8y,ATP is measured as the separation between the center of the arP doublet and central component of the BP triplet. The 31P chemical shifts are expressed with reference to 85% H,Po, as external standard.
A 31P NMR spectrum of MgATP (i.e., ATP with a saturating level of Mg2+) and the detailed temperature dependence of the quantity SrjATP at pH 7.2 (ionic strength - 0.15 M) and 40.5 MHz are shown in Figs. 1 and 2, respectively. The temperature readings for Fig. 2 were obtained by using a thermocouple inserted in the sample itself. The magnitude of the temperature dependence is 0.012 ppm/“C (i.e., 0.5 HzK at 40.5 MHz and 2.4 Hzl”C at 202.5 MHz 31P NMR frequency). It is therefore possible to measure sample temperature in highfield spectrometers using the MgATP thermometer with an accuracy better than O.Y’C, which is adequate for most biological NMR research. The exact origin of the observed temperature dependence of the S!J!jATPis not known but it arises predominantly from a shift of the ,BP resonance. Since the chemical shift of the BP resonance of ATP is sensitive to the conformation
T (“Cl FIG. 2. Temperature dependence of the quantity SkyATP(pH = 7.2; ~1 = 0.15 M).
COMMUNICATIONS
589
of the polyphosphate chain, the temperature dependence of the 8z$“TP probably reflects a changing conformation or state of the metal ion chelation of the polyphosphate chain. It should be emphasized that since, in the MgATP thermometer, only the difference in chemical shift of two resonances in a single spectrum is measured, the actual measurement is easier than measurements based on absolute chemical shifts. REFERENCES E. KOLLIE, R. L. ANDERSON, J. L. HORTON, AND M. J. ROBERTS, Rev. Sci. 48, 501 (1977). 2. I. Y. SLONNIM, B. M. ARSHAVA, AND V. N. KLYUCHNIKOV, Zh. Fiz. Khim. (1976) [Russ. J. Phys. Chem. 50, No. 1 (1976)]. 3. D. R. VIDRINE AND P. E. PETERSON, Anal. Chem. 48, 1301 (1976). 4. S. COMBRISSON AND T. PRANCE, J. Magn. Reson. 19, 108 (1975). 5. H. J. SCHNEIDER: W. FREITAY, AND M. SCHOMMER, J. Magn. Reson. 18, 393 (1975). 6. J. BORNAIS AND S. BROWNSTEIN, J. Magn. Reson. 29, 207 (1978). 7. G. C. LEVY, J. T. BAILEY, AND D. A. WRIGHT, J. Magn. Reson..37,* 353 (1980). 8. R. K. GUPTA, J. L. BENOVIC, AND Z. B. ROSE, J. Biol. Chem. 253, 6172 (1978). 9. R. K. GUPTA AND W. D. YUSHOK, Proc. Nat. Acad. Sci. USA 77, 2487 (1980). 10. R. K. GUPTA AND R. D. MOORE, J. Biol. Chem. 255, 3987 (1980). I.
T.
RAJ
K.
Instrum. 50, 277
GUPTA?
PRATIMA
GUPTA
The Institute for Cancer Research Fox Chase Cancer Center Philadelphia, Pennsylvania 19111 Received May 6, 1980
+ Research AM-0023 1).
Career
Development
Awardee
of the
United
States
Public
Health
Service
(NIH
OF MAGNETIC
RESONANCE
40, 587-589
(1980)
A Magnesium(II)ATP Thermometer for 31PNMR Studies of Biological Systems * A common problem in 31P NMR studies of biological systems is accurate measurement of sample temperature. Reliable temperature measurements require either a thermocouple placed in the sample itself (or in a sample of similar geometry and dielectric constant) or the use of a suitable compound having a temperature-dependent chemical shift. Difficulties in placing a thermocouple in the NMR sample tube, especially with superconducting spectrometers, and the lack of suitable compounds having temperature-dependent shielding characteristics for every nucleus studied have resulted in the use of a thermocouple placed either just below or above the receiver/transmitter coil for the measurement of flow gas temperature which may not be the same as the sample temperature. Such temperature readings become especially unreliable when high-power decoupling is used, which is usually the case with 31P NMR studies. There is also some evidence of errors in thermocouple measurements resulting from superconducting magnetic fields (1). Several lH, 2H, lgF, 13C, and 5gCo thermometers have been proposed previously (2-7). To use these existing nucleus-specific thermometers in 31P NMR studies, one needs to switch back and forth between the 31P nucleus and the thermometer nucleus for temperature measurements. This operation generally requires probe retuning and is inconvenient for variable-temperature operation even with broadband design NMR probes. Because of the widespread use of 31P NMR in biological research, it is desirable to have an NMR thermometer based on the 31P nucleus for convenient measurement of sample temperature in biological applications of 31P NMR. In this communication we propose a MgATP thermometer for 31P N studies. In the course of our investigations on the state of ionized magnesium in intact cells by the noninvasive 31P NMR spectroscopy (8-IO), we have found that the chemical shift difference between the aP and pP resonances in neutral pH solutions of MgATP (6f#ATP), which is independent of ionic strength and small variations in pH, shows a sizable temperature dependence. Since 31P resonances of ATP are sharp and narrow (51 Hz), and ATP is very soluble in aqueous medium to permit the attainment of a good signal-to-noise ratio in a few minutes of time averaging, it is possible to measure the chemical shift difference @$ATP with an accuracy of a few tenths of a hertz. Our own measurements of this difference on a given MgATP sample at 40.5 MHz (on a Varian XL-100) using 8000 data points and a spectral width of 1000 Hz are reproducible to 5 kO.2 Hz. * This work has been supported by NIH Grants AM-19454 RR-05539 to the Institute for Cancer Research from the appropriation from the Commonwealth of Pennsylvania.
and AM-13351, National Institutes
by Grants CA-06927 and of Health, and by an
587 0022-2364/X0/120587-03$02.00!0 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
588
COMMUNICATIONS
5
IO I5 Chemical Shift (ppm)
io
FIG. 1. A 31P NMR spectrum showing the aP, /3P, and yP resonances of MgATP at 40.5 MHz (pH 7.2, p = 0.15 M, and T = 37°C). The sample contained 10 mM ATP and 30 m/M MgC12. The quantity 8y,ATP is measured as the separation between the center of the arP doublet and central component of the BP triplet. The 31P chemical shifts are expressed with reference to 85% H,Po, as external standard.
A 31P NMR spectrum of MgATP (i.e., ATP with a saturating level of Mg2+) and the detailed temperature dependence of the quantity SrjATP at pH 7.2 (ionic strength - 0.15 M) and 40.5 MHz are shown in Figs. 1 and 2, respectively. The temperature readings for Fig. 2 were obtained by using a thermocouple inserted in the sample itself. The magnitude of the temperature dependence is 0.012 ppm/“C (i.e., 0.5 HzK at 40.5 MHz and 2.4 Hzl”C at 202.5 MHz 31P NMR frequency). It is therefore possible to measure sample temperature in highfield spectrometers using the MgATP thermometer with an accuracy better than O.Y’C, which is adequate for most biological NMR research. The exact origin of the observed temperature dependence of the S!J!jATPis not known but it arises predominantly from a shift of the ,BP resonance. Since the chemical shift of the BP resonance of ATP is sensitive to the conformation
T (“Cl FIG. 2. Temperature dependence of the quantity SkyATP(pH = 7.2; ~1 = 0.15 M).
COMMUNICATIONS
589
of the polyphosphate chain, the temperature dependence of the 8z$“TP probably reflects a changing conformation or state of the metal ion chelation of the polyphosphate chain. It should be emphasized that since, in the MgATP thermometer, only the difference in chemical shift of two resonances in a single spectrum is measured, the actual measurement is easier than measurements based on absolute chemical shifts. REFERENCES E. KOLLIE, R. L. ANDERSON, J. L. HORTON, AND M. J. ROBERTS, Rev. Sci. 48, 501 (1977). 2. I. Y. SLONNIM, B. M. ARSHAVA, AND V. N. KLYUCHNIKOV, Zh. Fiz. Khim. (1976) [Russ. J. Phys. Chem. 50, No. 1 (1976)]. 3. D. R. VIDRINE AND P. E. PETERSON, Anal. Chem. 48, 1301 (1976). 4. S. COMBRISSON AND T. PRANCE, J. Magn. Reson. 19, 108 (1975). 5. H. J. SCHNEIDER: W. FREITAY, AND M. SCHOMMER, J. Magn. Reson. 18, 393 (1975). 6. J. BORNAIS AND S. BROWNSTEIN, J. Magn. Reson. 29, 207 (1978). 7. G. C. LEVY, J. T. BAILEY, AND D. A. WRIGHT, J. Magn. Reson..37,* 353 (1980). 8. R. K. GUPTA, J. L. BENOVIC, AND Z. B. ROSE, J. Biol. Chem. 253, 6172 (1978). 9. R. K. GUPTA AND W. D. YUSHOK, Proc. Nat. Acad. Sci. USA 77, 2487 (1980). 10. R. K. GUPTA AND R. D. MOORE, J. Biol. Chem. 255, 3987 (1980). I.
T.
RAJ
K.
Instrum. 50, 277
GUPTA?
PRATIMA
GUPTA
The Institute for Cancer Research Fox Chase Cancer Center Philadelphia, Pennsylvania 19111 Received May 6, 1980
+ Research AM-0023 1).
Career
Development
Awardee
of the
United
States
Public
Health
Service
(NIH