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Aqueous shift reagents for high-resolution cationic nuclear magnetic resonance
JOURNAL
OF MAGNETIC
RESONANCE
46, 348-353 (1982)
Aqueous Shift Reagents for High-Resolution Magnetic Resonance
Cationic Nuclear
MARTIN M. PIKE AND CHARLES S. SPRINGER, JR.* Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794 Received November 3, 1981
In our efforts to study the transport of metal cations across biological membranes by nuclear magnetic resonance (NMR) (Z), we have realized the importance of directly monitoring physiological cations themselves. Modern NMR developments have made the consideration of routine studies of the magnetic isotopes of Na+, K+, Mg’+, and Ca’+ feasible (2). Examples of recent biological applications of such studies are: “Na+ (3, 4), 39K+ (5), 25Mg2+(6, 7a), and 43Ca2+(7). In order to realize the full potential of NMR in distinguishing mechanisms of transmembrane transport, it is necessary to render the resonance frequency of the cationic nucleus different (anisochronous) on one side of the membrane from the other (I). We report here some preliminary work in the development of aqueous shift reagents for cationic NMR and, in two separate communications, examples of their use in studying Na+ transport in model membrane vesicles (8), and in living cells (9). It seemed clear that, in order to maximize interaction with the observed metal cation, .water soluble anionic complexes of paramagnetic metal ions were required. This reasoning was first proffered by Elgavish and Reuben for aqueous shift reagents to be used with organic cations. They used the ethylenediaminetetraacetate ( EDTA4-) chelate of Pr 3+, Pr( EDTA)), to shift the ‘H resonancesof the pyridinium cation and the various methyl-substituted ammonium cations (10). Subsequently the analogous relaxation reagent, Gd(EDTA)-, was used to relax the resonances of “Na+ and 7Li+ (4). Anionic complexes of Gd3+ with EDTA4-, other polyaminocarboxylate ligands, nitrilotriacetate (NTA’-), and dipicolinate (DPA’), have also been used to relax the “N and 13C resonances of water soluble amines (II, 12). The Dy3+ complex of diethylenetriaminepentaacetate (DTPA’-) has been used as a relaxation reagent for glycine, alanine, and lactate by employing its effects on bulk magnetic susceptibility (13). We decided to test various shift reagents using 23Na NMR, the most amenable of those of the physiological cations (2). After initially disappointing results with Fe(CN)63- (no shifts)’ and Pr( EDTA)- (very small shifts), we decided to try com* To whom correspondence should be addressed. ’The anion Fe(CN)63- does act as a shift reagent for the tetramethylammonium ion (14), and for phosphatidylcholine membrane surfaces (15, 16, 17). However, we suspect that this is because of a chaotropic contribution to the interaction (18) not enjoyed by Na+ (19). Presumably, the utility of the relaxation reagent Cr(CN)63- for membrane surfaces (15), arises for similar reasons. 0022-2364/82/020348-06gO2.00/0 Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
348
349
COMMUNICATIONS
I -I
1
0
I
I
1
3 A (ppm)
4
5
I
I
2
1
6
7
b. -
FIG. 1. The 23Na+ NMR spectra (132.3 MHz, 11.75 T) of mixtures of NaCl (2.0 mM) and (HTEA),Dy(NTA)r in 50% DsO. The stoichiometric concentrations of (HTEA),Dy(NTA)r are: (a) 0 rn,W, (b) 0.50 mM, (c) 2.0 mM, (d) 8.0.mM, (e) 25.0 m&f, and (f) 50.0 mM. The temperature was approximately 297 K. For each spectrum, the number of free-induction decays accumulated was 128 and the total acquisition time was 52.5 sec. The vertical scales differ. The spectrometer was fieldfrequency locked on the solvent *H resonance which thus served as an internal reference. This is an imperfect correction for bulk susceptibility shifts if this resonance experiences any isotropic hype&e shift.
plexes of Dy3+, the paramagnetic lanthanide which usually produces the largest isotropic hyperfine shifts (20, 21). We found significant shifts with Dy(EDTA)-, but even greater ones using the Dy( DPA)33- and DY(NTA)*~- complexes, probably a result of the greater charge. Most of our recent work has employed the latter complex because of its greater solubility (22), and the slightly larger shifts it produces. The shift reagent chelate was prepared in situ according to the reaction l/2 Dy,03 + 2H3NTA + 3 TEA - 3HTEA+ + DY(NTA)~~- + 3/2H20,
[ 1]
where HTEA+ is HN(CHzCHzOH)s+. Figure 1 shows Z3Na NMR spectra (132.3 MHz, Bruker WM-500) of 2 m M NaCl in the presence of varying amounts of (HTEA)3Dy(NTA)2. Experimental details are given in the figure caption. The upfield isotropic hyperfine shift, A, is clearly observed. The line appears to retain a Lorentzian shape as it is shifted (indicating that the spectrum remains in the “fast motional narrowing” condition (23)). Some broadening induced by the shift reagent is observed but it is small compared to the shift. Figure 2 depicts typical hyperfine shift titration curves obtained at different (constant) NaCl concentrations. The continuous nature of these curves indicates that the equilibrium interaction of Na+ with DY(NTA)*~- responsible for the shift Na+ + DY(NTA)~~- P Na+ . DY(NTA)~~-
[21
is labile on the NMR timescale. Their shapes correspond to a weak interaction. The detailed nature of this binding is not known at this time: presumably, it involves
350
COMMUNICATIONS
632mM No* 5. P : : +;
:
:
:
8mM p #,’ ,/’ ,#,’ : ,’
fi
0
No+
/
/ .’,’ .,’,,C’ ,’
5
,..’
,A
/-.’
/
*.I-
2 mM Na*se.--O */-
/.d” ,.*’
JO
15
20
25
P FIG. 2. The isotropic hyperfine shift, A (23Na+ NMR) versus the stoichiometric mol ratio of shift reagent to Na+, p. The data include those in Fig. 1. The spectral parameters were the same as those of Fig. 1. The dashed curves are intended merely to guide the eye.
some type of ion-pairing (IO); probably solvent separated. On the basis of approximate graphical analyses, we suspect that, along with the 1:l stoichiometry shown in Eq. [2], 2:l (Na+:shift reagent) stoichiometry is important, that the apparent 1: 1 concentration binding constant is > 10 M-‘, and that the limiting shift is > 10 ppm. Accurate ion-pair stoichiometry, thermodynamic association constants, and limiting shifts can be determined by careful fitting of NMR binding isotherms (correcting for electrostatic effects on activity coefficients) (24). We are pursuing this although the situation is complicated by pH and countercation competition effects. We have varied the shift reagent countercation out of concern for possible effects of competition for the DY(NTA)~~- between the cation and Na+. (Elgavish and Reuben report two series with regard to competition for Pr(EDTA)-: Cs+ < Rb+ < NH,+ < Li+ < trimethylammonium and Cs+ < Rb+ < NH4+ < Li+ < methylammonium (IO).) Under our conditions, three different cations (HTEA+, HTRIS+(HaNC(CH20H)S’), and Li+) compete to quite similar extents. Also in this vein, we have noted that the observed hyperfine shift decreases at low pH values, being almost zero at pH = 2.3. Analogous trends in the effect of
351
COMMUNICATIONS
ki
a I b
-I
o
I
2 A @pm)
3
4
5
6
EL-
FIG. 3. The “Na+ NMR spectra (26.5 MHz, 2.35 T) of mixtures of NaCl (99 m&f), LilDy(NTA)2, and Lu(NO,), in 50% DZO. The stoichiometric concentrations of Li,Dy(NTA)Z and Lu(NO,), are: (a) 0 and 0 mA4, (b) 5 and 0 mM, (c) 10 and 0 mM, (d) 20 and 0 mM, (e) 40 and 0 mM, (f) 75 and 0 mM; (g) 40 and 5 mM, (h) 40 and 10 mM, (i) 40 and 20 mM, (j) 40 and 30 mM, and (k) 40 and 40 mM. The temperature was approximately 303 K. For each spectrum, the number of free-induction decays accumulated was 500 and the total acquisition time was 3.33 min. The solvent *H resonance served as an internal reference in the same sense as described for Fig. 1.
pH on the relaxation of the 23Na+ and 7Li+ resonances by Gd(EDTA)- have been reported (4). In many membrane transport studies, one m ight want shift reagent present only inside closed vesicles. The easiest way to accomplish this is to prepare the vesicles in the presence of the shift reagent anions and then to selectively deactivate only the outside shift reagent (8). We have found that deactivation can be simply achieved by titration of the shift reagent with the diamagnetic Lu’+ ion. This is clearly seen in Fig. 3, where the shifts of the 23Na+ resonance (26.5 MHz, Varian XL-100) of solutions 99 m M in NaCl produced by increasing concentrations of Li3Dy(NTA)2 (a through f) are reversed by increasing amounts of Lu3+ (added as the nitrate) (g through k). Experimental details are given in the figure caption. The Lu3+ deactivates the DY(NTA)~~-, presumably through the ligand exchange reactions: Lu’+ + DY(NTA)~~- Tr?Lu(NTA)~~(‘-‘) + DY(NTA)~-~~(+‘); Neither Dy(NTA) for Na+.
i = 1,2.
[3]
(i = 1) nor Dy3+ (i = 2) would be an effective shift reagent
352
COMMUNICATIONS
An interesting observation is made in comparing and contrasting the spectra shown in Fig. 1 (132.3 MHz, 11.74 T) and Fig. 3 (26.5 MHz, 2.35 T). Broadening induced by the shift reagent (in frequency units) is not appreciably dependent upon the magnetic field strength. This probably indicates that this additional relaxation has significant contributions from fluctuations in hyperfine dipolar interactions (20) and possibly from fluctuating electric field gradients at the 23Na+ nucleus attributable to the short lifetimes of the ion pairs. The 23Na+ nucleis (I = 3/2) has an electric quadrupole moment. The latter mechanism would not be properly termed “hyperfine.” It is quite likely that the anionic Dy3+ complexes reported here can be used as shift reagents for any of the magnetic cationic nuclei mentioned above. We have recently observed shifts of the ‘Li+ resonance induced by DY(DPA)~~- and DY(NTA)*~- (25). In contrast with the 23Na+ results, the DY(DPA)~~- complex induces larger shifts. The ion-pair interactions with *‘Mg*+ and 43Ca2+are likely to be stronger because of the increased charge on the cation. Another possible application of these shift reagents should be mentioned. The measurement of the magnitude of the binding of physiological metal cations to biological polyelectrolytes (proteins, nucleic acids, charged membrane surfaces) is an important and difficult problem (3, 6). We have recently elaborated a very sensitive NMR indicator approach to such binding (25, 26). An analogous method, employing these shift reagents, should be quite feasible. ACKNOWLEDGMENTS We thank the National Science Foundation (Grant PCM78-07918) for support of this work. We also thank Mr. Dennis Sorce for the initial experiments with Pr(EDTA)- and Mr. Reynold Homan for the initial suggestion of Dy(DPA),‘-. Our gratitude also goes to Mr. James Balschi and the staff of the Southern California Regional NMR facility at Cal Tech (Messrs. Thomas Perkins, Utpal Banerjee, Dr. William R. Croasmun, and Professor Sunney I. Chan) for their generous aid and hospitality in the use of the Bruker WM-500 (funded by NSF Grant CHE 79-16324). REFERENCES 1. D. Z. TING, P. S. HAGAN, S. I. CHAN, J. D. DOLL, AND C. S. SPRINGER, Biophys. J. 34, 189 (1981). 2. S. FOR&N, “Proceedings, European Conference on NMR of Macromolecules” (F. Conti, Ed.), p. 243, Lerici, Sassari/Sardenia, 1978. 3. M. L. BLEAM, C. F. ANDERSON, AND M. T. RECORD, Prof. Nat. Acad. Sci. USA 77,3085 (1980). 4. H. DEGANI AND G. A. ELGAVISH, FEBS Lett. 90, 357 (1978). 5. R. G. BRYANT, B&hem. Biophys. Res. Commun. 40, 1162 (1970). 6. D. M. ROSE, M. L. BLEAM, M. T. RECORD, AND R. G. BRYANT, Proc. Nat. Acad. Sci. USA 77, 6289 (1980). 7. (a) T. ANDERSSON, T. DRAKENBERG, S. FOR&N, AND E. THULIN, FEBS Let?. 125, 39 (1981). (b) T. ANDERSSON, T. DRAKENBERG, S. FOR&N, T. WIELOCH, AND M. LINDSTR~M, FEBS Lett. 123, 115 (1981). 8. M. M. PIKE, S. R. SIMON, J. A. BALXHI, AND C. S. SPRINGER, Proc. Nat. Acad. Sci. USA 79, (1982). 9. J. A. BALXHI, V. P. CIRILLO, AND C. S. SPRINGER, submitted for publication. 10. G. A. ELGAVISH AND J. REUBEN, J. Am. Chem. Sot. 99, 1762 (1977). Il. J. J. DECHTER AND G. C. LEVY, J. Magn. Reson. 30, 207 (1980). 12. T. J. WENZEL, M. E. ASHLEY, AND R. E. SIEVERS, Submitted for publication.
COMMUNICATIONS
353
13. K. M. BRINDLE, F. F. BROWN, I. D. CAMPBELL, C. GRATHWOHL,AND P. W. KUCHEL, Biochem. J. 180, 37 (1979). 14. M. P. N. GENT AND J. H. PRESTEGARD,B&him. Biophys. Acta 426, 17 (1976). 15. H. HAUSER, M. C. PHILLIPS, B. A. LEVINE, AND R. J. P. WILLIAMS, Nature (London) 261, 390
(1976). 16. R. J. KOSTELNIK AND S. M. CASTELLANO, J. Magn. Reson. 7, 219 (1972). 17. J. A. BERDEN, R. W. BARKER, AND G. H. RADDA, Biochim. Biophys. Acta 375, 186 (1975). 18. J. GRANDJEAN, unpublished results. 19. P. LASZLO AND A. STOKIS, J. Am. Chem. Sot. 102, 7818 (1980). 20. F. INAGAKI AND T. MIYAZAWA, Progr. NMR Spectrosc. 14, 67 (1981). 22. C. C. BRYDEN, C. N. REILLEY,AND J. F. DESREUX, Anal. Chem. 53, 1418 (1981). 22. H. DONATO AND R. B. MARTIN, J. Am. Chem. Sot. 94, 4129 ( 1972). 23. M. M. CIVAN AND M. SHPORER in “Biological Magnetic Resonance” (L. J. Berliner and J. Reuben,
Eds.), pp. l-32, Plenum, New York, 1978. 24. A. CHRZESZCZYK, A. WISHNIA, AND C. S. SPRINGER, Biochim. Biophys. Acta 648, 28 (1981). 25. J. A. BALSCHI, unpublished results. 26. A. CHRZESZCZYK, A. WISHNIA, AND C. S. SPRINGER, ACS Symp. 34,483 (1976).
OF MAGNETIC
RESONANCE
46, 348-353 (1982)
Aqueous Shift Reagents for High-Resolution Magnetic Resonance
Cationic Nuclear
MARTIN M. PIKE AND CHARLES S. SPRINGER, JR.* Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794 Received November 3, 1981
In our efforts to study the transport of metal cations across biological membranes by nuclear magnetic resonance (NMR) (Z), we have realized the importance of directly monitoring physiological cations themselves. Modern NMR developments have made the consideration of routine studies of the magnetic isotopes of Na+, K+, Mg’+, and Ca’+ feasible (2). Examples of recent biological applications of such studies are: “Na+ (3, 4), 39K+ (5), 25Mg2+(6, 7a), and 43Ca2+(7). In order to realize the full potential of NMR in distinguishing mechanisms of transmembrane transport, it is necessary to render the resonance frequency of the cationic nucleus different (anisochronous) on one side of the membrane from the other (I). We report here some preliminary work in the development of aqueous shift reagents for cationic NMR and, in two separate communications, examples of their use in studying Na+ transport in model membrane vesicles (8), and in living cells (9). It seemed clear that, in order to maximize interaction with the observed metal cation, .water soluble anionic complexes of paramagnetic metal ions were required. This reasoning was first proffered by Elgavish and Reuben for aqueous shift reagents to be used with organic cations. They used the ethylenediaminetetraacetate ( EDTA4-) chelate of Pr 3+, Pr( EDTA)), to shift the ‘H resonancesof the pyridinium cation and the various methyl-substituted ammonium cations (10). Subsequently the analogous relaxation reagent, Gd(EDTA)-, was used to relax the resonances of “Na+ and 7Li+ (4). Anionic complexes of Gd3+ with EDTA4-, other polyaminocarboxylate ligands, nitrilotriacetate (NTA’-), and dipicolinate (DPA’), have also been used to relax the “N and 13C resonances of water soluble amines (II, 12). The Dy3+ complex of diethylenetriaminepentaacetate (DTPA’-) has been used as a relaxation reagent for glycine, alanine, and lactate by employing its effects on bulk magnetic susceptibility (13). We decided to test various shift reagents using 23Na NMR, the most amenable of those of the physiological cations (2). After initially disappointing results with Fe(CN)63- (no shifts)’ and Pr( EDTA)- (very small shifts), we decided to try com* To whom correspondence should be addressed. ’The anion Fe(CN)63- does act as a shift reagent for the tetramethylammonium ion (14), and for phosphatidylcholine membrane surfaces (15, 16, 17). However, we suspect that this is because of a chaotropic contribution to the interaction (18) not enjoyed by Na+ (19). Presumably, the utility of the relaxation reagent Cr(CN)63- for membrane surfaces (15), arises for similar reasons. 0022-2364/82/020348-06gO2.00/0 Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
348
349
COMMUNICATIONS
I -I
1
0
I
I
1
3 A (ppm)
4
5
I
I
2
1
6
7
b. -
FIG. 1. The 23Na+ NMR spectra (132.3 MHz, 11.75 T) of mixtures of NaCl (2.0 mM) and (HTEA),Dy(NTA)r in 50% DsO. The stoichiometric concentrations of (HTEA),Dy(NTA)r are: (a) 0 rn,W, (b) 0.50 mM, (c) 2.0 mM, (d) 8.0.mM, (e) 25.0 m&f, and (f) 50.0 mM. The temperature was approximately 297 K. For each spectrum, the number of free-induction decays accumulated was 128 and the total acquisition time was 52.5 sec. The vertical scales differ. The spectrometer was fieldfrequency locked on the solvent *H resonance which thus served as an internal reference. This is an imperfect correction for bulk susceptibility shifts if this resonance experiences any isotropic hype&e shift.
plexes of Dy3+, the paramagnetic lanthanide which usually produces the largest isotropic hyperfine shifts (20, 21). We found significant shifts with Dy(EDTA)-, but even greater ones using the Dy( DPA)33- and DY(NTA)*~- complexes, probably a result of the greater charge. Most of our recent work has employed the latter complex because of its greater solubility (22), and the slightly larger shifts it produces. The shift reagent chelate was prepared in situ according to the reaction l/2 Dy,03 + 2H3NTA + 3 TEA - 3HTEA+ + DY(NTA)~~- + 3/2H20,
[ 1]
where HTEA+ is HN(CHzCHzOH)s+. Figure 1 shows Z3Na NMR spectra (132.3 MHz, Bruker WM-500) of 2 m M NaCl in the presence of varying amounts of (HTEA)3Dy(NTA)2. Experimental details are given in the figure caption. The upfield isotropic hyperfine shift, A, is clearly observed. The line appears to retain a Lorentzian shape as it is shifted (indicating that the spectrum remains in the “fast motional narrowing” condition (23)). Some broadening induced by the shift reagent is observed but it is small compared to the shift. Figure 2 depicts typical hyperfine shift titration curves obtained at different (constant) NaCl concentrations. The continuous nature of these curves indicates that the equilibrium interaction of Na+ with DY(NTA)*~- responsible for the shift Na+ + DY(NTA)~~- P Na+ . DY(NTA)~~-
[21
is labile on the NMR timescale. Their shapes correspond to a weak interaction. The detailed nature of this binding is not known at this time: presumably, it involves
350
COMMUNICATIONS
632mM No* 5. P : : +;
:
:
:
8mM p #,’ ,/’ ,#,’ : ,’
fi
0
No+
/
/ .’,’ .,’,,C’ ,’
5
,..’
,A
/-.’
/
*.I-
2 mM Na*se.--O */-
/.d” ,.*’
JO
15
20
25
P FIG. 2. The isotropic hyperfine shift, A (23Na+ NMR) versus the stoichiometric mol ratio of shift reagent to Na+, p. The data include those in Fig. 1. The spectral parameters were the same as those of Fig. 1. The dashed curves are intended merely to guide the eye.
some type of ion-pairing (IO); probably solvent separated. On the basis of approximate graphical analyses, we suspect that, along with the 1:l stoichiometry shown in Eq. [2], 2:l (Na+:shift reagent) stoichiometry is important, that the apparent 1: 1 concentration binding constant is > 10 M-‘, and that the limiting shift is > 10 ppm. Accurate ion-pair stoichiometry, thermodynamic association constants, and limiting shifts can be determined by careful fitting of NMR binding isotherms (correcting for electrostatic effects on activity coefficients) (24). We are pursuing this although the situation is complicated by pH and countercation competition effects. We have varied the shift reagent countercation out of concern for possible effects of competition for the DY(NTA)~~- between the cation and Na+. (Elgavish and Reuben report two series with regard to competition for Pr(EDTA)-: Cs+ < Rb+ < NH,+ < Li+ < trimethylammonium and Cs+ < Rb+ < NH4+ < Li+ < methylammonium (IO).) Under our conditions, three different cations (HTEA+, HTRIS+(HaNC(CH20H)S’), and Li+) compete to quite similar extents. Also in this vein, we have noted that the observed hyperfine shift decreases at low pH values, being almost zero at pH = 2.3. Analogous trends in the effect of
351
COMMUNICATIONS
ki
a I b
-I
o
I
2 A @pm)
3
4
5
6
EL-
FIG. 3. The “Na+ NMR spectra (26.5 MHz, 2.35 T) of mixtures of NaCl (99 m&f), LilDy(NTA)2, and Lu(NO,), in 50% DZO. The stoichiometric concentrations of Li,Dy(NTA)Z and Lu(NO,), are: (a) 0 and 0 mA4, (b) 5 and 0 mM, (c) 10 and 0 mM, (d) 20 and 0 mM, (e) 40 and 0 mM, (f) 75 and 0 mM; (g) 40 and 5 mM, (h) 40 and 10 mM, (i) 40 and 20 mM, (j) 40 and 30 mM, and (k) 40 and 40 mM. The temperature was approximately 303 K. For each spectrum, the number of free-induction decays accumulated was 500 and the total acquisition time was 3.33 min. The solvent *H resonance served as an internal reference in the same sense as described for Fig. 1.
pH on the relaxation of the 23Na+ and 7Li+ resonances by Gd(EDTA)- have been reported (4). In many membrane transport studies, one m ight want shift reagent present only inside closed vesicles. The easiest way to accomplish this is to prepare the vesicles in the presence of the shift reagent anions and then to selectively deactivate only the outside shift reagent (8). We have found that deactivation can be simply achieved by titration of the shift reagent with the diamagnetic Lu’+ ion. This is clearly seen in Fig. 3, where the shifts of the 23Na+ resonance (26.5 MHz, Varian XL-100) of solutions 99 m M in NaCl produced by increasing concentrations of Li3Dy(NTA)2 (a through f) are reversed by increasing amounts of Lu3+ (added as the nitrate) (g through k). Experimental details are given in the figure caption. The Lu3+ deactivates the DY(NTA)~~-, presumably through the ligand exchange reactions: Lu’+ + DY(NTA)~~- Tr?Lu(NTA)~~(‘-‘) + DY(NTA)~-~~(+‘); Neither Dy(NTA) for Na+.
i = 1,2.
[3]
(i = 1) nor Dy3+ (i = 2) would be an effective shift reagent
352
COMMUNICATIONS
An interesting observation is made in comparing and contrasting the spectra shown in Fig. 1 (132.3 MHz, 11.74 T) and Fig. 3 (26.5 MHz, 2.35 T). Broadening induced by the shift reagent (in frequency units) is not appreciably dependent upon the magnetic field strength. This probably indicates that this additional relaxation has significant contributions from fluctuations in hyperfine dipolar interactions (20) and possibly from fluctuating electric field gradients at the 23Na+ nucleus attributable to the short lifetimes of the ion pairs. The 23Na+ nucleis (I = 3/2) has an electric quadrupole moment. The latter mechanism would not be properly termed “hyperfine.” It is quite likely that the anionic Dy3+ complexes reported here can be used as shift reagents for any of the magnetic cationic nuclei mentioned above. We have recently observed shifts of the ‘Li+ resonance induced by DY(DPA)~~- and DY(NTA)*~- (25). In contrast with the 23Na+ results, the DY(DPA)~~- complex induces larger shifts. The ion-pair interactions with *‘Mg*+ and 43Ca2+are likely to be stronger because of the increased charge on the cation. Another possible application of these shift reagents should be mentioned. The measurement of the magnitude of the binding of physiological metal cations to biological polyelectrolytes (proteins, nucleic acids, charged membrane surfaces) is an important and difficult problem (3, 6). We have recently elaborated a very sensitive NMR indicator approach to such binding (25, 26). An analogous method, employing these shift reagents, should be quite feasible. ACKNOWLEDGMENTS We thank the National Science Foundation (Grant PCM78-07918) for support of this work. We also thank Mr. Dennis Sorce for the initial experiments with Pr(EDTA)- and Mr. Reynold Homan for the initial suggestion of Dy(DPA),‘-. Our gratitude also goes to Mr. James Balschi and the staff of the Southern California Regional NMR facility at Cal Tech (Messrs. Thomas Perkins, Utpal Banerjee, Dr. William R. Croasmun, and Professor Sunney I. Chan) for their generous aid and hospitality in the use of the Bruker WM-500 (funded by NSF Grant CHE 79-16324). REFERENCES 1. D. Z. TING, P. S. HAGAN, S. I. CHAN, J. D. DOLL, AND C. S. SPRINGER, Biophys. J. 34, 189 (1981). 2. S. FOR&N, “Proceedings, European Conference on NMR of Macromolecules” (F. Conti, Ed.), p. 243, Lerici, Sassari/Sardenia, 1978. 3. M. L. BLEAM, C. F. ANDERSON, AND M. T. RECORD, Prof. Nat. Acad. Sci. USA 77,3085 (1980). 4. H. DEGANI AND G. A. ELGAVISH, FEBS Lett. 90, 357 (1978). 5. R. G. BRYANT, B&hem. Biophys. Res. Commun. 40, 1162 (1970). 6. D. M. ROSE, M. L. BLEAM, M. T. RECORD, AND R. G. BRYANT, Proc. Nat. Acad. Sci. USA 77, 6289 (1980). 7. (a) T. ANDERSSON, T. DRAKENBERG, S. FOR&N, AND E. THULIN, FEBS Let?. 125, 39 (1981). (b) T. ANDERSSON, T. DRAKENBERG, S. FOR&N, T. WIELOCH, AND M. LINDSTR~M, FEBS Lett. 123, 115 (1981). 8. M. M. PIKE, S. R. SIMON, J. A. BALXHI, AND C. S. SPRINGER, Proc. Nat. Acad. Sci. USA 79, (1982). 9. J. A. BALXHI, V. P. CIRILLO, AND C. S. SPRINGER, submitted for publication. 10. G. A. ELGAVISH AND J. REUBEN, J. Am. Chem. Sot. 99, 1762 (1977). Il. J. J. DECHTER AND G. C. LEVY, J. Magn. Reson. 30, 207 (1980). 12. T. J. WENZEL, M. E. ASHLEY, AND R. E. SIEVERS, Submitted for publication.
COMMUNICATIONS
353
13. K. M. BRINDLE, F. F. BROWN, I. D. CAMPBELL, C. GRATHWOHL,AND P. W. KUCHEL, Biochem. J. 180, 37 (1979). 14. M. P. N. GENT AND J. H. PRESTEGARD,B&him. Biophys. Acta 426, 17 (1976). 15. H. HAUSER, M. C. PHILLIPS, B. A. LEVINE, AND R. J. P. WILLIAMS, Nature (London) 261, 390
(1976). 16. R. J. KOSTELNIK AND S. M. CASTELLANO, J. Magn. Reson. 7, 219 (1972). 17. J. A. BERDEN, R. W. BARKER, AND G. H. RADDA, Biochim. Biophys. Acta 375, 186 (1975). 18. J. GRANDJEAN, unpublished results. 19. P. LASZLO AND A. STOKIS, J. Am. Chem. Sot. 102, 7818 (1980). 20. F. INAGAKI AND T. MIYAZAWA, Progr. NMR Spectrosc. 14, 67 (1981). 22. C. C. BRYDEN, C. N. REILLEY,AND J. F. DESREUX, Anal. Chem. 53, 1418 (1981). 22. H. DONATO AND R. B. MARTIN, J. Am. Chem. Sot. 94, 4129 ( 1972). 23. M. M. CIVAN AND M. SHPORER in “Biological Magnetic Resonance” (L. J. Berliner and J. Reuben,
Eds.), pp. l-32, Plenum, New York, 1978. 24. A. CHRZESZCZYK, A. WISHNIA, AND C. S. SPRINGER, Biochim. Biophys. Acta 648, 28 (1981). 25. J. A. BALSCHI, unpublished results. 26. A. CHRZESZCZYK, A. WISHNIA, AND C. S. SPRINGER, ACS Symp. 34,483 (1976).