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Sodium-23 NMR of intact bovine lens and vitreous humor
JOURNAL
OF MAGNETIC
RESONANCE
63, 439-444 (1985)
Sodium-23 NMR of Intact Bovine Lens and Vitreous Humor JAY W. PETTEGREW,” THOMAS GLONEK,~ NANCY J. MINSHEW,* AND DONALD E. WOESSNER~ *Laboratory of Neurophysics, Department of Psychiatry, Western Psychiatric Institute and Clinic, University of Pittsburgh, Pittsburgh, Pennsylvania 15213; TN&ear Magnetic Resonance Laboralory, Chicago College of Osteopathic Medicine, Chicago, Illinois 60615; and @Jobi Research and Development Corporation, DRD, Dallas, Texas 75234 Received August 27, 1984; revised December 6, 1984 Sodium-23 nuclear magnetic resonance was used to investigate the chemical functionality of sodium ion in the intact, crystalline bovine lens and vitreous humor, as compared to physiologic saline. The NMR parameters of chemical shift and TI and T2 relaxation are consistent with the interpretation that the chemical functionality of sodium ion in bovine vitreous humor is identical with that of sodium ion in physiologic salt solution. Identical studies on intact bovine lens demonstrated that some fraction of the sodium ion in the lens is relatively immobilized. The calculated sodium ion interaction lifetime in the crystalline bovine lens is 3 X 1O-g s, which is qualitatively similar to interactions described between sodium ion and the proteins troponin C and calmodulin. 0 1985 Academic PIW, 1~.
Translocation of sodium ion across biomembranes is of fundamental importance to cellular physiology. This process is clearly dependent on the physiologic integrity of the living cell as well as the chemical functionality of the sodium ion. The advantage of a noninvasive, nonperturbing technique for monitoring tissue sodium flux is obvious. Such an advantage is afforded by NMR spectroscopy, a well documented technique for analysis of in vivo and ex vivo metabolism (I). Sodium23 NMR spectroscopy provides direct quantitation of tissue sodium content as well as qualitative information on the chemical functionality of sodium ions in living systems. We have recently demonstrated in human erythrocytes that intracellular sodium ion concentration determined by sodium-23 NMR correlated very well with flame photometry determinations. These studies further demonstrated that some fraction of the intracellular sodium ion was relatively immobilized (2). In the present study sodium-23 NMR was used to measure sodium ion dynamics in the physiologically intact bovine crystalline lens and vitreous humor relative to a physiologic saline reference solution. Lens and vitreous humor extraction was accomplished by sag&al and frontal incisions at the posterior pole of the optic globe. These incisions were extended to the ora serrata using curved blunt scissors. The vitreous humor was separated from the lens with a glass spatula and collected as large gel fragments in a 50 ml beaker. The zonules were then cut with curved scissors, and the lens removed with a glass lens loupe. Excess vitreous humor was removed from the lens with the aid of a 439
0022-2364185 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
440
PETTEGREW
ET AL.
dissection microscope. The data presented are the results of studies on eight separate bovine eyes. A modified physiologic Earle’s buffer (3) at pH 7.4 was prepared by replacing NaCl with sucrose to yield an isotonic solution: glucose, 5.6 mlM; KCl, 5.4 mM; CaCL, 1.8 mM, MgCl*, 1.4 mM; tris(hydroxymethyl)aminomethane, 10.0 mM; sucrose, 262 mM, 298 mOsm. Samples were positioned in 20 mm NMR sample tubes so as to be in the center of the spectrometer’s receiver/transmitter coil. This was accomplished by placing the sample on a gauze support immersed in buffer. The lens was placed in the sample tube with the anterior surface down and the lens long axis perpendicular to the sample tube long axis. Changing the angle of the lens axis relative to the sample tube axis did not alter the sodium resonance parameters. Sodium-23 NMR analysis was performed using a Nicolet NT-200 (4.7 T) widebore NMR spectrometer operating at 52.92 MHz for sodium-23. The sodium-23 spectrometer probe was a 20 mm fixed-frequency device constructed of quartz instead of glass to avoid baseline distortion due to the sodium silicates ordinarily present in glass. The pulse sequences used were one pulse, T, by inversion-recovery, and T2 by the Carr-Purcell-Meiboom-Gill sequence, modified to use composite 180” pulses (4). The experimental parameters used were 90” pulse, 50.75 ps; 180” pulse, 10 1.50 ps; acquisition time, 0.8 1 s; 8 192 data points per free induction decay; pulse delay, 1.0 s; number of acquisitions, 100-200; sweep width, k2500 Hz. All studies were conducted at 23 + 1°C nonspinning. In our studies, high-resolution linewidths of a spin-4 nuclide, such as phosphorus-3 1, with nonspinning 20 mm samples are less than 2 Hz and are usually approximately 1 Hz. A solution of 0.1 M NaCl in distilled, deionized water was used as the chemical shift (6) reference and was designated 0.006. This solution gave a single resonance peak with a linewidth at half height (zQ,J of 14.6 Hz for a nonspinning 20 mm sample and v1/2 = 6.1 Hz for the same sample spinning at 24 rps (Table 1). The sodium resonance of lens in Earle’s modified buffer was centered at 0.126 with v1/2 = 25.6 Hz. Bovine vitreous humor also gave a single resonance centered at 0.076 with vi/z = 20.7 Hz. The 0.1 M NaCl reference solution gave a sodium-23 T, and T2 of 60 ms which is in agreement with data previously reported (2, 5). The Tl and T2 of the vitreous humor was also 60 ms. The Tl of the lens, however, was 37 ms (Fig. 1) and the T2
TABLE 1 NMR Chemical Shift (6, ppm) and Relaxation Parameters for the Indicated Samples Sample
Chemical shift in 6 (pw)
VII2 Wd
Tl C-4
T2 b-4
0.1 M NaCl Lens (in Earle’s buffer) Vitreous humor
0.00 0.12 0.07
14.6 25.6 20.7
60 37.8 f 1.0 60.9 rk 0.9
60 28.2 + 1.6 60.3 f 4.4
Notes. The T, and T2 values for bovine lens and vitreous humor are given as the mean + SD (N = 8).
SODIUM-23
4
NMR
2
0
OF
BOVINE
EYE
-2
-4
-6 PPM
0.6
0.8
1.0 SEC
441
I 0.0
0.2
0.4
1. T, by inversion-recovery for bovine lens in buffer. (a) Stacked plot for different values of time delays (7) between the 180 and 90” pulses. (b) Integrated peak area versus 7 values. FIG.
was 28 ms (Fig. 2), both values being markedly shorter than those of sodium ion in vitreous humor or sodium ion in simple aqueous solution. High-resolution sodium-23 spectra can be obtained from intact tissue and other biologic samples using the noninvasive, nonperturbing technique of sodium-23 NMR spectroscopy. The key observations in this study pertain to the differences in chemical functionality of sodium ion as it relates to tissue type. Chemical functionality is reflected in chemical-shift and relaxation-time parameters. Sodium ion in vitreous humor has chemical-shift and relaxation properties essentially identical to those of sodium ion in aqueous solution, implying a similar chemical state and suggesting that sodium ion in vitreous humor is coordinated with water molecules just as it is in simple aqueous solutions. The linewidth of vitreous humor is wider (20.7 Hz) than that of 0.1 M NaCl (14.6 Hz), but the T, and T2 values are the same (60 ms) in both tissues. Some explanation is needed for this apparent discrepancy. Contributions to linewidth broadening are generally of two types: sample characteristics or instrumental conditions. Sample characteristics include line-broadening mechanisms associated with reduced atomic/molecular motion (chemical-shift anisotropy, dipolar, scalar, quadrupolar, or spin-rotational couplings). These mechanisms are reflected by a shortening of both Tl and Tg (as compared to the extreme narrowing condition)
442
PETTEGREW
ET AL.
A.
~‘.1.“,“‘,‘~‘,“‘,.~.,...,~~~,~.~,..~
0.0
0.2
0.4 0.6
0.8
1.0
1.2
1.4
1.6 1.8
2.0 SEC
FIG. 2. T2 by Carr-Purcell-Meiboom-Gill sequence modified to use composite 180” pulses for bovine lens in buffer. (a) Stacked plot of resonance peaks for different delay (7) values. (b) Integrated resonance peak area versus T values.
but with T2 shortened more than Tl. Instrumental conditions which can give rise to artificially broadened linewidths are generally related to inhomogeneities in Ho and/or Hi. Inhomogeneities in the E&, XY plane across the sample can be reduced by sample spinning, while inhomogeneities in Hi can be reduced by using composite 180” pulses (4). In the present study, sample magnetic susceptibility problems probably contribute to some of the sample linewidths. For example, a 0.1 M NaCl solution gave a substantially narrower linewidth under spinning conditions (6.1 Hz spinning; 14.6 Hz nonspinning) indicating Ho inhomogeneity. In preliminary Tl and T, experiments on 0.1 M NaCl, it was observed that Ti by inversion-recovery was approximately 11 to 2 times the value of T2 obtained by a Carr-PurcellMeiboom-Gill (CPMG) sequence even though the extreme narrowing condition should apply. This was interpreted as indicating Hi inhomogeneity. Repeat Tz experiments using a CPMG sequence containing composite 180” pulses yielded Tz values equal to T, (60 ms each) confirming the presence of Hi inhomogeneity which had been corrected by composite 180” pulses. It is therefore essential that appropriate pulse sequences be performed when attempting to measure T, and T, values in biological samples. In particular, T2'sderived from linewidth measurements may be very deceptive for these reasons. Clearly, the broadened vitreous humor linewidth is not due to restricted atomic/molecular motion but is most likely due to No and Hi inhomogeneities related to the bulk magnetic susceptibility of the sample.
SODIUM-23
NMR OF BOVINE
EYE
443
The sodium chemical-shift characteristics of bovine crystalline lens are essentially identical to those of sodium ion in aqueous solution, but in contrast, the relaxation times are strikingly shortened. The predominant mechanism for sodium NMR relaxation is electric quadrupolar which involves an interaction at the sodium nucleus between the sodium nuclear electric quadrupole moment and the electrostatic field gradient. This interaction is directly proportional to the square of the electrostatic field gradient and also to the spectral energy density which is a function of the electrostatic field gradient motion (5, 6). The electrostatic field gradient is the vector sum of the electric charge distribution (from other ionic charges and charged coordination sites) and the electric dipole moments (from the solvation water molecules). The spectral energy density is expressed in terms of correlation times, 7=, which statistically describe the average directional lifetime of the different contributions to the electrostatic field gradient. If there are different sodium ion environments, the net sodium relaxation is a function of the magnitudes of the electrostatic field gradients and their associated correlation times. In interpreting sodium-23 relaxation of sodium ions in aqueous solutions, it is customary to assume extreme narrowing conditions. In the extreme narrowing condition the correlation time associated with a given electrostatic field gradient contribution is less than the reciprocal of the nuclear magnetic resonance frequency. Under this condition, a change in only the magnitude of the electrostatic field gradient causes equal percentage changes in the relaxation rates l/T, and 1/T2, regardless of the correlation time values. When all of the correlation times remain within bounds of the extreme narrowing condition (7, + 3 X IO-’ s for the spectrometer used in the present studies) a change in only 7c will also cause equal changes in 1/T, and l/T, and T, = T2. In simple aqueous solution T, N 10-r l s, which is well within the bounds of the extreme narrowing condition. However, if a correlation time becomes sufficiently long (7, >2 3 X lo-’ s for the 52.92 MHz NMR frequency), then T, becomes greater than T2. In intact crystalline bovine lens, T, is in fact longer than T2. This indicates that a long correlation time (7, Z= 3 X lo-’ s) is involved in shortening the lens sodium relaxation times from the values obtained in simple aqueous solution. It also implies that some fraction of the sodium ion in lens is relatively immobilized (away from the extreme narrowing condition) as it would be if its coordination sites were occupied by protein functional groups rather than water molecules. From the data of Table 1, a sodium ionprotein interaction would have a lifetime on the order of 3 X low9 s, which is qualitatively similar to the interactions described between sodium ion and the proteins troponin C (7) and calmodulin (8). In summary, this study demonstrates the usefulness of NMR spectroscopy in the investigation of sodium chemical functionality in the mammalian lens. Alterations in the lens sodium ion chemical functionality can be expected in some disease states, Such an alteration is being sought in lens undergoing cataract formation. ACKNOWLEDGMENTS We thank Ms. Linda Raney and Ms. Connie Pollack for their secretarial assistance in preparing this manuscript. The work was supported in part by a grant from the United Cerebral Palsy Foundation.
444
PETTEGREW
ET
AL.
REFERENCES 1. T. GLONEK, in “Phosphorus Chemistry Directed Towards Biology” (W. J. Stec, Ed.), pp. 157-174, Pergamon, New York/Oxford, 1980. 2. J. W. PETTEGREW, D. E. WOESSNER, N. J. MINSHEW, AND T. GLONEK, J. Magn. Reson. 51, 185 (1984). 3. J. V. GREINER, S. J. KOPP, D. R. SANDERS, AND T. GLONEK, Invest. Ophthalmol. Vis. Sci. 21, 700 (1981). 4. R. FREEMAN, S. P. KEMPSELL, AND M. H. LEVITT, J. Magn. Reson. 38, 453 (1980). 5. D. C. CHANG AND D. E. WOESSNER, J. Magn. Reson. 30, 185 (1978). 6. P. S. HUBBARD, J. Chew Phys. 53, 985 (1970); T. E. BULL, J. Magn. Reson. 8, 344 (1972). 7. A. DELVILLE, J. GRANDJEAN, P. LASZLO, C. GERDAY, Z. GRABAREK, AND W. DRABIKOWSKI, Eur. J. Biochem. 105, 289 (1980). 8. A. DELVILLE, J. GRANDJEAN, P. LASZLO, C. GERDAY, H. BRZESKA, AND W. DRABIKOWSKI, Eur. J. Biochem. 109, 515 (1980).
OF MAGNETIC
RESONANCE
63, 439-444 (1985)
Sodium-23 NMR of Intact Bovine Lens and Vitreous Humor JAY W. PETTEGREW,” THOMAS GLONEK,~ NANCY J. MINSHEW,* AND DONALD E. WOESSNER~ *Laboratory of Neurophysics, Department of Psychiatry, Western Psychiatric Institute and Clinic, University of Pittsburgh, Pittsburgh, Pennsylvania 15213; TN&ear Magnetic Resonance Laboralory, Chicago College of Osteopathic Medicine, Chicago, Illinois 60615; and @Jobi Research and Development Corporation, DRD, Dallas, Texas 75234 Received August 27, 1984; revised December 6, 1984 Sodium-23 nuclear magnetic resonance was used to investigate the chemical functionality of sodium ion in the intact, crystalline bovine lens and vitreous humor, as compared to physiologic saline. The NMR parameters of chemical shift and TI and T2 relaxation are consistent with the interpretation that the chemical functionality of sodium ion in bovine vitreous humor is identical with that of sodium ion in physiologic salt solution. Identical studies on intact bovine lens demonstrated that some fraction of the sodium ion in the lens is relatively immobilized. The calculated sodium ion interaction lifetime in the crystalline bovine lens is 3 X 1O-g s, which is qualitatively similar to interactions described between sodium ion and the proteins troponin C and calmodulin. 0 1985 Academic PIW, 1~.
Translocation of sodium ion across biomembranes is of fundamental importance to cellular physiology. This process is clearly dependent on the physiologic integrity of the living cell as well as the chemical functionality of the sodium ion. The advantage of a noninvasive, nonperturbing technique for monitoring tissue sodium flux is obvious. Such an advantage is afforded by NMR spectroscopy, a well documented technique for analysis of in vivo and ex vivo metabolism (I). Sodium23 NMR spectroscopy provides direct quantitation of tissue sodium content as well as qualitative information on the chemical functionality of sodium ions in living systems. We have recently demonstrated in human erythrocytes that intracellular sodium ion concentration determined by sodium-23 NMR correlated very well with flame photometry determinations. These studies further demonstrated that some fraction of the intracellular sodium ion was relatively immobilized (2). In the present study sodium-23 NMR was used to measure sodium ion dynamics in the physiologically intact bovine crystalline lens and vitreous humor relative to a physiologic saline reference solution. Lens and vitreous humor extraction was accomplished by sag&al and frontal incisions at the posterior pole of the optic globe. These incisions were extended to the ora serrata using curved blunt scissors. The vitreous humor was separated from the lens with a glass spatula and collected as large gel fragments in a 50 ml beaker. The zonules were then cut with curved scissors, and the lens removed with a glass lens loupe. Excess vitreous humor was removed from the lens with the aid of a 439
0022-2364185 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
440
PETTEGREW
ET AL.
dissection microscope. The data presented are the results of studies on eight separate bovine eyes. A modified physiologic Earle’s buffer (3) at pH 7.4 was prepared by replacing NaCl with sucrose to yield an isotonic solution: glucose, 5.6 mlM; KCl, 5.4 mM; CaCL, 1.8 mM, MgCl*, 1.4 mM; tris(hydroxymethyl)aminomethane, 10.0 mM; sucrose, 262 mM, 298 mOsm. Samples were positioned in 20 mm NMR sample tubes so as to be in the center of the spectrometer’s receiver/transmitter coil. This was accomplished by placing the sample on a gauze support immersed in buffer. The lens was placed in the sample tube with the anterior surface down and the lens long axis perpendicular to the sample tube long axis. Changing the angle of the lens axis relative to the sample tube axis did not alter the sodium resonance parameters. Sodium-23 NMR analysis was performed using a Nicolet NT-200 (4.7 T) widebore NMR spectrometer operating at 52.92 MHz for sodium-23. The sodium-23 spectrometer probe was a 20 mm fixed-frequency device constructed of quartz instead of glass to avoid baseline distortion due to the sodium silicates ordinarily present in glass. The pulse sequences used were one pulse, T, by inversion-recovery, and T2 by the Carr-Purcell-Meiboom-Gill sequence, modified to use composite 180” pulses (4). The experimental parameters used were 90” pulse, 50.75 ps; 180” pulse, 10 1.50 ps; acquisition time, 0.8 1 s; 8 192 data points per free induction decay; pulse delay, 1.0 s; number of acquisitions, 100-200; sweep width, k2500 Hz. All studies were conducted at 23 + 1°C nonspinning. In our studies, high-resolution linewidths of a spin-4 nuclide, such as phosphorus-3 1, with nonspinning 20 mm samples are less than 2 Hz and are usually approximately 1 Hz. A solution of 0.1 M NaCl in distilled, deionized water was used as the chemical shift (6) reference and was designated 0.006. This solution gave a single resonance peak with a linewidth at half height (zQ,J of 14.6 Hz for a nonspinning 20 mm sample and v1/2 = 6.1 Hz for the same sample spinning at 24 rps (Table 1). The sodium resonance of lens in Earle’s modified buffer was centered at 0.126 with v1/2 = 25.6 Hz. Bovine vitreous humor also gave a single resonance centered at 0.076 with vi/z = 20.7 Hz. The 0.1 M NaCl reference solution gave a sodium-23 T, and T2 of 60 ms which is in agreement with data previously reported (2, 5). The Tl and T2 of the vitreous humor was also 60 ms. The Tl of the lens, however, was 37 ms (Fig. 1) and the T2
TABLE 1 NMR Chemical Shift (6, ppm) and Relaxation Parameters for the Indicated Samples Sample
Chemical shift in 6 (pw)
VII2 Wd
Tl C-4
T2 b-4
0.1 M NaCl Lens (in Earle’s buffer) Vitreous humor
0.00 0.12 0.07
14.6 25.6 20.7
60 37.8 f 1.0 60.9 rk 0.9
60 28.2 + 1.6 60.3 f 4.4
Notes. The T, and T2 values for bovine lens and vitreous humor are given as the mean + SD (N = 8).
SODIUM-23
4
NMR
2
0
OF
BOVINE
EYE
-2
-4
-6 PPM
0.6
0.8
1.0 SEC
441
I 0.0
0.2
0.4
1. T, by inversion-recovery for bovine lens in buffer. (a) Stacked plot for different values of time delays (7) between the 180 and 90” pulses. (b) Integrated peak area versus 7 values. FIG.
was 28 ms (Fig. 2), both values being markedly shorter than those of sodium ion in vitreous humor or sodium ion in simple aqueous solution. High-resolution sodium-23 spectra can be obtained from intact tissue and other biologic samples using the noninvasive, nonperturbing technique of sodium-23 NMR spectroscopy. The key observations in this study pertain to the differences in chemical functionality of sodium ion as it relates to tissue type. Chemical functionality is reflected in chemical-shift and relaxation-time parameters. Sodium ion in vitreous humor has chemical-shift and relaxation properties essentially identical to those of sodium ion in aqueous solution, implying a similar chemical state and suggesting that sodium ion in vitreous humor is coordinated with water molecules just as it is in simple aqueous solutions. The linewidth of vitreous humor is wider (20.7 Hz) than that of 0.1 M NaCl (14.6 Hz), but the T, and T2 values are the same (60 ms) in both tissues. Some explanation is needed for this apparent discrepancy. Contributions to linewidth broadening are generally of two types: sample characteristics or instrumental conditions. Sample characteristics include line-broadening mechanisms associated with reduced atomic/molecular motion (chemical-shift anisotropy, dipolar, scalar, quadrupolar, or spin-rotational couplings). These mechanisms are reflected by a shortening of both Tl and Tg (as compared to the extreme narrowing condition)
442
PETTEGREW
ET AL.
A.
~‘.1.“,“‘,‘~‘,“‘,.~.,...,~~~,~.~,..~
0.0
0.2
0.4 0.6
0.8
1.0
1.2
1.4
1.6 1.8
2.0 SEC
FIG. 2. T2 by Carr-Purcell-Meiboom-Gill sequence modified to use composite 180” pulses for bovine lens in buffer. (a) Stacked plot of resonance peaks for different delay (7) values. (b) Integrated resonance peak area versus T values.
but with T2 shortened more than Tl. Instrumental conditions which can give rise to artificially broadened linewidths are generally related to inhomogeneities in Ho and/or Hi. Inhomogeneities in the E&, XY plane across the sample can be reduced by sample spinning, while inhomogeneities in Hi can be reduced by using composite 180” pulses (4). In the present study, sample magnetic susceptibility problems probably contribute to some of the sample linewidths. For example, a 0.1 M NaCl solution gave a substantially narrower linewidth under spinning conditions (6.1 Hz spinning; 14.6 Hz nonspinning) indicating Ho inhomogeneity. In preliminary Tl and T, experiments on 0.1 M NaCl, it was observed that Ti by inversion-recovery was approximately 11 to 2 times the value of T2 obtained by a Carr-PurcellMeiboom-Gill (CPMG) sequence even though the extreme narrowing condition should apply. This was interpreted as indicating Hi inhomogeneity. Repeat Tz experiments using a CPMG sequence containing composite 180” pulses yielded Tz values equal to T, (60 ms each) confirming the presence of Hi inhomogeneity which had been corrected by composite 180” pulses. It is therefore essential that appropriate pulse sequences be performed when attempting to measure T, and T, values in biological samples. In particular, T2'sderived from linewidth measurements may be very deceptive for these reasons. Clearly, the broadened vitreous humor linewidth is not due to restricted atomic/molecular motion but is most likely due to No and Hi inhomogeneities related to the bulk magnetic susceptibility of the sample.
SODIUM-23
NMR OF BOVINE
EYE
443
The sodium chemical-shift characteristics of bovine crystalline lens are essentially identical to those of sodium ion in aqueous solution, but in contrast, the relaxation times are strikingly shortened. The predominant mechanism for sodium NMR relaxation is electric quadrupolar which involves an interaction at the sodium nucleus between the sodium nuclear electric quadrupole moment and the electrostatic field gradient. This interaction is directly proportional to the square of the electrostatic field gradient and also to the spectral energy density which is a function of the electrostatic field gradient motion (5, 6). The electrostatic field gradient is the vector sum of the electric charge distribution (from other ionic charges and charged coordination sites) and the electric dipole moments (from the solvation water molecules). The spectral energy density is expressed in terms of correlation times, 7=, which statistically describe the average directional lifetime of the different contributions to the electrostatic field gradient. If there are different sodium ion environments, the net sodium relaxation is a function of the magnitudes of the electrostatic field gradients and their associated correlation times. In interpreting sodium-23 relaxation of sodium ions in aqueous solutions, it is customary to assume extreme narrowing conditions. In the extreme narrowing condition the correlation time associated with a given electrostatic field gradient contribution is less than the reciprocal of the nuclear magnetic resonance frequency. Under this condition, a change in only the magnitude of the electrostatic field gradient causes equal percentage changes in the relaxation rates l/T, and 1/T2, regardless of the correlation time values. When all of the correlation times remain within bounds of the extreme narrowing condition (7, + 3 X IO-’ s for the spectrometer used in the present studies) a change in only 7c will also cause equal changes in 1/T, and l/T, and T, = T2. In simple aqueous solution T, N 10-r l s, which is well within the bounds of the extreme narrowing condition. However, if a correlation time becomes sufficiently long (7, >2 3 X lo-’ s for the 52.92 MHz NMR frequency), then T, becomes greater than T2. In intact crystalline bovine lens, T, is in fact longer than T2. This indicates that a long correlation time (7, Z= 3 X lo-’ s) is involved in shortening the lens sodium relaxation times from the values obtained in simple aqueous solution. It also implies that some fraction of the sodium ion in lens is relatively immobilized (away from the extreme narrowing condition) as it would be if its coordination sites were occupied by protein functional groups rather than water molecules. From the data of Table 1, a sodium ionprotein interaction would have a lifetime on the order of 3 X low9 s, which is qualitatively similar to the interactions described between sodium ion and the proteins troponin C (7) and calmodulin (8). In summary, this study demonstrates the usefulness of NMR spectroscopy in the investigation of sodium chemical functionality in the mammalian lens. Alterations in the lens sodium ion chemical functionality can be expected in some disease states, Such an alteration is being sought in lens undergoing cataract formation. ACKNOWLEDGMENTS We thank Ms. Linda Raney and Ms. Connie Pollack for their secretarial assistance in preparing this manuscript. The work was supported in part by a grant from the United Cerebral Palsy Foundation.
444
PETTEGREW
ET
AL.
REFERENCES 1. T. GLONEK, in “Phosphorus Chemistry Directed Towards Biology” (W. J. Stec, Ed.), pp. 157-174, Pergamon, New York/Oxford, 1980. 2. J. W. PETTEGREW, D. E. WOESSNER, N. J. MINSHEW, AND T. GLONEK, J. Magn. Reson. 51, 185 (1984). 3. J. V. GREINER, S. J. KOPP, D. R. SANDERS, AND T. GLONEK, Invest. Ophthalmol. Vis. Sci. 21, 700 (1981). 4. R. FREEMAN, S. P. KEMPSELL, AND M. H. LEVITT, J. Magn. Reson. 38, 453 (1980). 5. D. C. CHANG AND D. E. WOESSNER, J. Magn. Reson. 30, 185 (1978). 6. P. S. HUBBARD, J. Chew Phys. 53, 985 (1970); T. E. BULL, J. Magn. Reson. 8, 344 (1972). 7. A. DELVILLE, J. GRANDJEAN, P. LASZLO, C. GERDAY, Z. GRABAREK, AND W. DRABIKOWSKI, Eur. J. Biochem. 105, 289 (1980). 8. A. DELVILLE, J. GRANDJEAN, P. LASZLO, C. GERDAY, H. BRZESKA, AND W. DRABIKOWSKI, Eur. J. Biochem. 109, 515 (1980).