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Determination of magnesium binding to macromolecules
ANALYTICAL
BIOCHEMISTRY
160,468-470
Determination
(1987)
of Magnesium
Binding to Macromolecules’
DEAN L. OLSON, DAVID W. DEERFIELD II, POLA BERKOWITZ, RICHARD G. HISKEY,~ AND LEE G. PEDERSEN Department
of Chemistry,
The University
of North
Carolina
at Chapel
Hill,
North
Carolina
27514
Received July 23, 1986 An equilibrium dialysis technique for examining magnesium binding to macromolecules is described. The technique is used to determine the binding constants of magnesium to human prothrombin. This procedure should be of great utility for many biochemical systems which exhibit magnesium affinity. 0 1987 Academic press, Inc. KEY WORDS: prothrombin; magnesium binding; equilibrium dialysis; coagulation; y-carboxyglutamic acid.
Metal ions play a mediating role in numerous biological processes. Divalent metal ions, particularly Mg(II), are effective in stabilizing the double helix structure of DNA (1). Ca(II), on the other hand, is required in the coagulation cascade for both the formation of the active enzyme complex (e.g., the prothrombinase complex is a combination of Factors Xa (enzyme) and Va (coenzyme), an acidic phospholipid surface, and Ca(I1)) and for the interaction of the substrate (prothrombin in this case) with the activated enzyme complex (2,3). Mg(I1) ions alone will not support the coagulation cascade; however, low levels of Mg(II), in the presence of Ca(II), can exhibit a synergistic effect (3). Muscle contraction also involves both divalent metal ions; Mg(I1) is a cofactor for the necessary ATPase, but ATP hydrolysis with subsequent muscle contraction proceeds only if Ca(I1) is also present (1). The understanding of processes involving metal ions is frequently facilitated by an accurate metal ion binding determination over the range of the physiological response.
Ca(I1) binding can be effectively evaluated by the use of 45Ca, an inexpensive, commercially available radionuclide with a 164 day half-life (3-5). Mg(I1) does not have a convenient isotope, and consequently, most Mg(I1) determinations have relied on techniques such as atomic absorption (6,7) or divalent ion-selective electrode (8), or on less direct methods provided by calorimetry (6), calorimetry (9), or fluorometric methods (10). When the macromolecule is not a strong metal ion binder (calmodulin (11) would be considered a very strong binder), radionuelides provide the most reliable metal ion binding data. The physiological roles of Ca(I1) and Mg(I1) appear to be very different. In our studies of metal ion binding to several coagulation proteins, it became necessary to develop an equilibrium dialysis procedure utilizing a radionuclide of Mg(I1) to compliment the previously determined Ca(I1) ion binding data. This note reports a Mg(I1) equilibrium dialysis procedure based on 28Mg (tv2 = 21 h) with an application to the binding of Mg(I1) by human prothrombin. Prothrombin contains 10 y-carboxyglutamyl residues (2) in the N-terminal region. The malonate side chains of these residues are responsible for the Ca(I1) and Mg(I1) site
’ This work was supported by Grants HL-27995 (L.G.P.) and HL-20161 (R.G.H.) from the National Institutes of Health, U.S. Public Health Service. * To whom correspondence should be addressed. 0003-2697187 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
468
EQUILIBRIUM
DIALYSIS
DETERMINATION
binding constants on the order of 1O3M-‘, an intermediate binding value. EXPERIMENTAL
OF MAGNESIUM 7000
469
BINDING
-
PROCEDURES
Human prothrombin was isolated and purified (12) and used at a concentration of 20 WM (1.43 mg ml-’ with EAFo= 13.8, M, = 72,000, A%$ = A;& - 1.706 . A@) (3,12). Equilibrium dialysis was performed using 10 cells (pH 7.50, 0.10 M NaCl, 0.02 M Tris, 25.OO”C, 2-ml Teflon cells), rotated at 20 rpm (Spectrum Medical, Los Angeles, CA), and equilibrated for 24 h. Fast-equilibrating disk membranes were employed after thorough cleaning (5) (Spectra/Par 2, 12- 14,000 MWCO, 47 mm). Highest purity commercially available reagents were used throughout. All solutions were made with deionized, distilled water (~5 ppb heavy metals, Hydro, Inc., Research Triangle Park, NC). Dialysis solutions were handled using Hamilton (Reno, NV) glass syringes with Teflon-tipped plungers and Teflon needles. **Mg obtained from Brookhaven National Laboratory, New York, was used as a radioactive tracer for Mg(I1). Each dialysis cell was spiked with Mg(I1) (7.6 PM) originating from the **Mg solution. After equilibration, four aliquots (100 ~1) were removed from each cell side and each sample was put in a glass scintillation vial with Na,EDTA (0.5 ml, 50 mM) and Fisher Scinti Verse II ( 10 ml). After vigorous mixing, samples were counted to a 2a cpm error of 0.1% in a 10-2000 keV region with a Packard 300CD liquid scintillation counter. Since a 28Mg liquid scintillation standard has never been produced and a calibration curve would be virtually impossible to generate, it was not possible to correct for counting efficiency. A 100% recovery of Mg(I1) was therefore assumed in each cell. The presence of protein in the vial did not affect counting efficiency. The decay of **Mg in a scintillation vial prepared identically to a sample was monitored for 5 days and an effective half-life was calculated (tl/, = 1304 min, r = - 1.0000).
- 4coo-i c/7 30002000
.
-
‘oooL----ix 1234567
B FIG. 1. Scatchard plot from the equilibrium dialysis data of human prothrombin (20 PM) with Mg(I1). The line represents the Langmuir loading of seven equal affinity, noninteracting sites with a site binding constant of 962 M-l. V = [Mg] b,,d/[prothrombin], S = ?/[Mg]r=.
This experimentally obtained value was used to correct the counts per minute for the amount of decay that occurred while the samples were counted. This procedure was equivalent to counting the eight aliquots from each titration cell at the same time. If any of the 4 corrected cpm values for a given cell half was outside the 2a confidence interval of the other three, it was not used. At least 3 cpm values were averaged for each cell half. RESULTS
The Mg(I1) equilibrium dialysis binding data is represented as a Scatchard plot (13) in Fig. 1. The data were analyzed ( 14,15) using the NLIN option of the SAS (16) package that utilizes the DUD (doesn’t use derivatives) procedure (17). A reasonable fit was obtained using a simple Langmuir loading equation assuming seven equal affinity, noninteracting sites ( 18) with a site binding constant of 962 M-‘. A fit of the thermodynamic Adair equation (19) assuming seven sites gave Ki (M-l), i = 1 to 7: 3829, 7875, 1000, 763, 500, 3 19, 100. These results are dramatically different than those found for the Ca(I1) binding to similar proteins; for Ca(II), a very pronounced concave downward Scat-
OLSON ET AL.
470
chard plot representing enhanced cooperativity for the binding of the second and third stoichiometric Ca(I1) has been reported for bovine prothrombin (5). The Mg(II) method described here should be of considerable utility for a wide range of experimental biochemical studies. ACKNOWLEDGMENTS We thank Bryan Neal, Dougald M. Monroe, and James B. Meade for a generous amount of human prothrombin.
REFERENCES 1. Hughes, M. N. (198 1) The Inorganic Chemistry of Biological Processes, 2nd ed., Wiley, New York. 2. Suttie, J. W., and Jackson, C. M. (1977) Physiol. Rev. 57, l-70. 3. Prendergast, F. G., and Mann, K. G. (1978) J. Biol. Chem. 252,840-850. 4. Johnson, A. E., Esmon, N. L., Laue, T. M., and Esmon, C. T. (1983) J. Biol. Chem. 258, 5554-5560. 5.
Deerfield, D. W., II, Berkowitz, P., Olson, D. L., Wells, S., Hoke, R. A., Koehler, K. A., Pedersen,
L. G., and Hiskey, R. G. (1986) J. Biol. Chem. 261,4833-4839.
6. Margalit, R., and Schejter, A. (1974) Eur. J. Biothem. 46,387-39 1. 7. Moeschler, H. J., Schaer, J., and Cox, J. A. (1980) Eur. J. Biochem. 111,73-78. 8. Sander, C., and Ts’o, P. 0. P. (197 1) J. Mol. Biol. 55, 1. 9. Rialdi, G., Levy, J., and Biltonen, R. (1972) Biochemistry 11, 2472-2479. 10. Bryant, D. T. W. (1985) Biochem. J. 226,613-616. 11. Cheung, W. Y. (1980) Science 207, 19-27. 12. Kisiel, W., and Hanahan, D. J. (1973) Biochim. Biophys. Acta 304, 103- 113. 13. Klotz, I. M. (1986) Introduction to Biomolecular Energetics, Academic Press, New York, p. 114. 14. Deerheld, D. W., II, Olson, D. L., Berkowitz, P., Byrd, P. A., Koehler, K. A., Pedersen, L. G., and Hiskey, R. G., submitted for publication. 15. Perlmutter-Hayman, B. (1986) Act. Chem. Rex 19, 90-96.
16. SAS Institute, Caty, North Carolina. 17. Ralston, M. L., and Jennrich, R. I. ( 1979) Technometrics 1, 7- 14. 18. Langmuir, I. (1916) J. Amer. Chem. Sot. 38, 2221-2295; (1918) J. Amer. Chem. Sot. 40, 1361-1403. 19. Adair, G. S. (1925) J. Biol. Chem. 63, 529-545.
BIOCHEMISTRY
160,468-470
Determination
(1987)
of Magnesium
Binding to Macromolecules’
DEAN L. OLSON, DAVID W. DEERFIELD II, POLA BERKOWITZ, RICHARD G. HISKEY,~ AND LEE G. PEDERSEN Department
of Chemistry,
The University
of North
Carolina
at Chapel
Hill,
North
Carolina
27514
Received July 23, 1986 An equilibrium dialysis technique for examining magnesium binding to macromolecules is described. The technique is used to determine the binding constants of magnesium to human prothrombin. This procedure should be of great utility for many biochemical systems which exhibit magnesium affinity. 0 1987 Academic press, Inc. KEY WORDS: prothrombin; magnesium binding; equilibrium dialysis; coagulation; y-carboxyglutamic acid.
Metal ions play a mediating role in numerous biological processes. Divalent metal ions, particularly Mg(II), are effective in stabilizing the double helix structure of DNA (1). Ca(II), on the other hand, is required in the coagulation cascade for both the formation of the active enzyme complex (e.g., the prothrombinase complex is a combination of Factors Xa (enzyme) and Va (coenzyme), an acidic phospholipid surface, and Ca(I1)) and for the interaction of the substrate (prothrombin in this case) with the activated enzyme complex (2,3). Mg(I1) ions alone will not support the coagulation cascade; however, low levels of Mg(II), in the presence of Ca(II), can exhibit a synergistic effect (3). Muscle contraction also involves both divalent metal ions; Mg(I1) is a cofactor for the necessary ATPase, but ATP hydrolysis with subsequent muscle contraction proceeds only if Ca(I1) is also present (1). The understanding of processes involving metal ions is frequently facilitated by an accurate metal ion binding determination over the range of the physiological response.
Ca(I1) binding can be effectively evaluated by the use of 45Ca, an inexpensive, commercially available radionuclide with a 164 day half-life (3-5). Mg(I1) does not have a convenient isotope, and consequently, most Mg(I1) determinations have relied on techniques such as atomic absorption (6,7) or divalent ion-selective electrode (8), or on less direct methods provided by calorimetry (6), calorimetry (9), or fluorometric methods (10). When the macromolecule is not a strong metal ion binder (calmodulin (11) would be considered a very strong binder), radionuelides provide the most reliable metal ion binding data. The physiological roles of Ca(I1) and Mg(I1) appear to be very different. In our studies of metal ion binding to several coagulation proteins, it became necessary to develop an equilibrium dialysis procedure utilizing a radionuclide of Mg(I1) to compliment the previously determined Ca(I1) ion binding data. This note reports a Mg(I1) equilibrium dialysis procedure based on 28Mg (tv2 = 21 h) with an application to the binding of Mg(I1) by human prothrombin. Prothrombin contains 10 y-carboxyglutamyl residues (2) in the N-terminal region. The malonate side chains of these residues are responsible for the Ca(I1) and Mg(I1) site
’ This work was supported by Grants HL-27995 (L.G.P.) and HL-20161 (R.G.H.) from the National Institutes of Health, U.S. Public Health Service. * To whom correspondence should be addressed. 0003-2697187 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
468
EQUILIBRIUM
DIALYSIS
DETERMINATION
binding constants on the order of 1O3M-‘, an intermediate binding value. EXPERIMENTAL
OF MAGNESIUM 7000
469
BINDING
-
PROCEDURES
Human prothrombin was isolated and purified (12) and used at a concentration of 20 WM (1.43 mg ml-’ with EAFo= 13.8, M, = 72,000, A%$ = A;& - 1.706 . A@) (3,12). Equilibrium dialysis was performed using 10 cells (pH 7.50, 0.10 M NaCl, 0.02 M Tris, 25.OO”C, 2-ml Teflon cells), rotated at 20 rpm (Spectrum Medical, Los Angeles, CA), and equilibrated for 24 h. Fast-equilibrating disk membranes were employed after thorough cleaning (5) (Spectra/Par 2, 12- 14,000 MWCO, 47 mm). Highest purity commercially available reagents were used throughout. All solutions were made with deionized, distilled water (~5 ppb heavy metals, Hydro, Inc., Research Triangle Park, NC). Dialysis solutions were handled using Hamilton (Reno, NV) glass syringes with Teflon-tipped plungers and Teflon needles. **Mg obtained from Brookhaven National Laboratory, New York, was used as a radioactive tracer for Mg(I1). Each dialysis cell was spiked with Mg(I1) (7.6 PM) originating from the **Mg solution. After equilibration, four aliquots (100 ~1) were removed from each cell side and each sample was put in a glass scintillation vial with Na,EDTA (0.5 ml, 50 mM) and Fisher Scinti Verse II ( 10 ml). After vigorous mixing, samples were counted to a 2a cpm error of 0.1% in a 10-2000 keV region with a Packard 300CD liquid scintillation counter. Since a 28Mg liquid scintillation standard has never been produced and a calibration curve would be virtually impossible to generate, it was not possible to correct for counting efficiency. A 100% recovery of Mg(I1) was therefore assumed in each cell. The presence of protein in the vial did not affect counting efficiency. The decay of **Mg in a scintillation vial prepared identically to a sample was monitored for 5 days and an effective half-life was calculated (tl/, = 1304 min, r = - 1.0000).
- 4coo-i c/7 30002000
.
-
‘oooL----ix 1234567
B FIG. 1. Scatchard plot from the equilibrium dialysis data of human prothrombin (20 PM) with Mg(I1). The line represents the Langmuir loading of seven equal affinity, noninteracting sites with a site binding constant of 962 M-l. V = [Mg] b,,d/[prothrombin], S = ?/[Mg]r=.
This experimentally obtained value was used to correct the counts per minute for the amount of decay that occurred while the samples were counted. This procedure was equivalent to counting the eight aliquots from each titration cell at the same time. If any of the 4 corrected cpm values for a given cell half was outside the 2a confidence interval of the other three, it was not used. At least 3 cpm values were averaged for each cell half. RESULTS
The Mg(I1) equilibrium dialysis binding data is represented as a Scatchard plot (13) in Fig. 1. The data were analyzed ( 14,15) using the NLIN option of the SAS (16) package that utilizes the DUD (doesn’t use derivatives) procedure (17). A reasonable fit was obtained using a simple Langmuir loading equation assuming seven equal affinity, noninteracting sites ( 18) with a site binding constant of 962 M-‘. A fit of the thermodynamic Adair equation (19) assuming seven sites gave Ki (M-l), i = 1 to 7: 3829, 7875, 1000, 763, 500, 3 19, 100. These results are dramatically different than those found for the Ca(I1) binding to similar proteins; for Ca(II), a very pronounced concave downward Scat-
OLSON ET AL.
470
chard plot representing enhanced cooperativity for the binding of the second and third stoichiometric Ca(I1) has been reported for bovine prothrombin (5). The Mg(II) method described here should be of considerable utility for a wide range of experimental biochemical studies. ACKNOWLEDGMENTS We thank Bryan Neal, Dougald M. Monroe, and James B. Meade for a generous amount of human prothrombin.
REFERENCES 1. Hughes, M. N. (198 1) The Inorganic Chemistry of Biological Processes, 2nd ed., Wiley, New York. 2. Suttie, J. W., and Jackson, C. M. (1977) Physiol. Rev. 57, l-70. 3. Prendergast, F. G., and Mann, K. G. (1978) J. Biol. Chem. 252,840-850. 4. Johnson, A. E., Esmon, N. L., Laue, T. M., and Esmon, C. T. (1983) J. Biol. Chem. 258, 5554-5560. 5.
Deerfield, D. W., II, Berkowitz, P., Olson, D. L., Wells, S., Hoke, R. A., Koehler, K. A., Pedersen,
L. G., and Hiskey, R. G. (1986) J. Biol. Chem. 261,4833-4839.
6. Margalit, R., and Schejter, A. (1974) Eur. J. Biothem. 46,387-39 1. 7. Moeschler, H. J., Schaer, J., and Cox, J. A. (1980) Eur. J. Biochem. 111,73-78. 8. Sander, C., and Ts’o, P. 0. P. (197 1) J. Mol. Biol. 55, 1. 9. Rialdi, G., Levy, J., and Biltonen, R. (1972) Biochemistry 11, 2472-2479. 10. Bryant, D. T. W. (1985) Biochem. J. 226,613-616. 11. Cheung, W. Y. (1980) Science 207, 19-27. 12. Kisiel, W., and Hanahan, D. J. (1973) Biochim. Biophys. Acta 304, 103- 113. 13. Klotz, I. M. (1986) Introduction to Biomolecular Energetics, Academic Press, New York, p. 114. 14. Deerheld, D. W., II, Olson, D. L., Berkowitz, P., Byrd, P. A., Koehler, K. A., Pedersen, L. G., and Hiskey, R. G., submitted for publication. 15. Perlmutter-Hayman, B. (1986) Act. Chem. Rex 19, 90-96.
16. SAS Institute, Caty, North Carolina. 17. Ralston, M. L., and Jennrich, R. I. ( 1979) Technometrics 1, 7- 14. 18. Langmuir, I. (1916) J. Amer. Chem. Sot. 38, 2221-2295; (1918) J. Amer. Chem. Sot. 40, 1361-1403. 19. Adair, G. S. (1925) J. Biol. Chem. 63, 529-545.