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Complex formation equilibria of phosphocreatine with sodium, potassium and magnesium ions
Polyhedron 21 (2002) 1481 /1484 www.elsevier.com/locate/poly
Complex formation equilibria of phosphocreatine with sodium, potassium and magnesium ions Franco Cecconi a, Chiara Frassineti b, Peter Gans c, Stefano Iotti d, Stefano Midollini a, Antonio Sabatini e,*, Alberto Vacca e a I.S.S.E.C.C., Consiglio Nazionale delle Ricerche, I-50132 Firenze, Italy Dipartimento di Scienze Biomediche, Universita` di Modena, I-41100 Modena, Italy c School of Chemistry, University of Leeds, Leeds LS2 9JT, UK d Dipartimento di Medicina Clinica e Biotecnologia Applicata ‘D. Campanacci’, Universita` di Bologna, I-40138 Bologna, Italy e Dipartimento di Chimica, Universita` di Firenze, Via della Lastruccia, I-50019 Sesto Fiorentino, Italy b
Received 24 October 2001; accepted 15 November 2001
Abstract The formation of complexes between phosphocreatine, H2O3PNHC( /NH)N(CH3)CH2CO2H, and the ions Na , K and Mg2 have been investigated under physiological conditions (aqueous solution, T/37 8C and I /0.25 mol dm3) by means of 31P NMR spectroscopy. Only 1:1 complexes have been identified. Stability constants have been determined with the aid of the new computer program HYPNMR-2000. log10 K values were found to be/0.5(2),/0.3(2) and 1.43(3), respectively. The formation constant for the potassium complex is two orders of magnitude less that the literature value. # 2002 Published by Elsevier Science Ltd. Keywords: Phoshocreatine;
31
P NMR spectroscopy; Sodium complexes; Potassium complexes; Magnesium complexes
1. Introduction In the cells of tissues such as muscle and brain the high energy demand required for the cell function is satisfied by the ATP hydrolysis. The ATP is regenerated by the creatine kinase (CK) catalysed reaction (1) which is a magnesium-dependent transphosphorilation reaction. PCrADP 0 ATPCr
(1)
Cr stands for creatine, N -amidinosarcosine, and PCr for its N- phosphonic derivative, phosphocreatine.
* Corresponding author. Tel.: /39-055-475-3276; fax: /39-055354-845. E-mail address: [email protected] (A. Sabatini).
Each reagent in Eq. (1) should be understood [1,2] to include the various chemical species in the equilibrium system that contain it. For example, ATP is generally in equilibrium with its magnesium complex. The equilibrium constant of the reaction is usually reported in terms of the concentration of the free ionic forms. Since direct measurement of concentrations of the free ionic forms is not feasible, it is convenient to use an apparent ? , which is defined in terms of equilibrium constant, K CK ? the sums of the concentrations of all related forms. K CK is very useful operationally, but has the drawback of being dependent on both H and the concentrations of the principal metal ions which are present in the cytosolic medium, namely, Mg2, Na and K but not Ca2 whose influence can be neglected since, in vivo, it is almost completely bound to proteins such as calmodulin and calbinding D-proteins. To quantify this dependence the formation constants of all the species involved in the CK equilibrium with Mg2, Na and K need to be known. At physiological pH PCr is present as the anion PCr2 and the oxygen atoms on the phosphonate group can co-ordinate to the metal cations. Thus, PCr2 competes with other phosphate-
0277-5387/02/$ - see front matter # 2002 Published by Elsevier Science Ltd. PII: S 0 2 7 7 - 5 3 8 7 ( 0 2 ) 0 0 9 5 1 - 8
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F. Cecconi et al. / Polyhedron 21 (2002) 1481 /1484
containing species, such as HPO42, ATP and ADP, in forming metal-ion complexes. Few thermodynamic data have been reported for the formation of PCr complexes and those that have are so contradictory that it is stated that the published data do not meet the criteria required for a critical selection in the NIST database [3]. Formation constants have been published for MgPCr [4] and KPCr [5], but nothing has been reported for NaPCr . This is strange as the sodium and potassium complexes of ATP and ADP are usually quite similar [3,6]. Another disquieting aspect of the published data is the small difference between the formation constants of MgPCr and KPCr, since with analogous ligands magnesium complexes are much more stable than the corresponding potassium complexes [3,6]. Information regarding the stability of PCr complexes is essential for the determination of the concentrations of the species present in the cytosolic medium. We have, therefore, undertaken a systematic study, using 31P NMR spectroscopy, of complexation of the phosphate ligands PCr, ATP, ADP and AMP with the three cations Na , K e Mg2, under physiological conditions (T /37 8C and I /0.25 M). We now report the results for phosphocreatine.
2. Experimental 2.1. Chemicals Na2PCr ×/4.5H2O (Sigma) was used without further purification (Calc. for C4H17N3O9.5PNa2, C, 14.29; H, 5.10; N, 12.50. Found: C, 14.20; H, 5.21; N, 12.50%). The 31P NMR spectra of this substance show that hydrolysis is negligible in aqueous solution as the intensity of the phosphate ion signal was always less than 1% of the total integrated intensity. Analytical grade reagents (Merck) were used to prepare stock solutions of Me4NCl, NaCl, KCl and MgCl2 using weighed amounts of solute and solvent. D2O (99.8%, Merck) and reagent grade Bu4NCl (Fluka) and Bu4PCl (50% aqueous solution, Merck) were used as supplied. 2.2. NMR measurements 31
P{1H} NMR spectra were recorded at 81.02 MHz using a Bruker AC 200 spectrometer. The probe temperature was 37.09/0.2 8C. Field frequency stabilisation was established by using as solvent H2O /D2O (80:20 by volume). All chemical shifts are reported in ppm toward higher frequency relative to external 85% aqueous H3PO4. No corrections were made for differences in bulk susceptibly in the various samples. Digital resolution was 0.216 Hz per point. In all cases p/3 pulses were used. The feed was accumulated in 32 k data points
with zero filling to 64 k data points before Fourier transformation. The acquisition time was 4.62 s. 2.3. Spectra and compositions of the solutions used At least ten solutions were prepared for each system. Solutions were stored at approximately 4 8C to prevent hydrolysis. The concentration of Na2PCr was approximately 0.02 mol dm 3 in all solutions. The concentrations of the metal halides (NaCl, KCl and MgCl2) ranged between zero and the concentration corresponding to an ionic strength of 0.25 mol dm 3. Where necessary, the solutions were made upto constant ionic strength of 0.25 mol dm 3 by addition of Me4NCl for the NaCl /PCr and KCl /PCr systems or NaCl for the MgCl2 /PCr system. A further series of 11 replicate solutions in the NaCl /PCr system was prepared, but with Bu4PCl added as an internal reference and replacing Me4NCl by Bu4NCl. 2.4. Computational procedures In the course of this work the new computer program has been written and developed. This is a generalised program for the determination of stability constants from NMR chemical shift data in systems where exchange is rapid on the NMR time-scale. It has a novel structure. The main program is written in VISUAL BASIC and runs as a typical WINDOWS application. The main program contains extensive facilities for data input and preliminary calculations including the possibility of interactive data fitting with graphical displays of observed and calculated data. The stability constant refinement is a compiled FORTRAN module, called from the main program, which is based on the earlier HYPNMR program [7]. The refinement module reads data from an input file and records the results of the computation in an output file. On termination of the refinement the output file is automatically opened in the main program and is displayed in a customised viewer. HYPNMR-2000 can handle any number of individual resonances, including resonances from more than one nucleus, such as 13C and 1H, at the same time. There are no limitations on the number of reagents or stability constants that can be specified. Further details are available at http://www.chem.leeds.ac.uk/people/peter_gans/hypnmr.htm. HYPNMR-2000
3. Results and discussion All spectra showed a single narrow resonance, as illustrated in Fig. 1. This indicates that all the species related to phosphocreatine are in rapid equilibrium on the NMR time-scale. Confirmation of this state of affairs is provided by the smooth variation of the
F. Cecconi et al. / Polyhedron 21 (2002) 1481 /1484
Fig. 1. 31P NMR spectrum of a solution containing Na2PCr 0.020 mol dm 3; T/37 8C and I/0.25 mol dm 3, made up with Me4NCl.
chemical shift with changes in concentration of the metallic cations: with Na and K there is a slight drift to low field with increasing metal ion concentration and
1483
Fig. 3. Hyss distribution diagram of phosphocreatine as a function of pH. The pKa values were taken from Ref. [8].
with Mg2 there is a marked displacement to high field. These trends are illustrated in Fig. 2. Four protonation constants have been determined for phosphocreatine [8]. The three pKa values, /0.41, 2.84 and 4.46, were assigned to dissociation of one proton from the /PO3H2 group, dissociation of the carboxyl group proton and dissociation of a second proton from the phosphate group, respectively. The fourth pKa value, 11.2, relates to further deprotonation of PCr2. A diagram showing the distribution of the species as a function of pH is shown in Fig. 3. This diagram, produced by the program HYSS-2000 [9], shows that PCr2 is the only species present between pH 7 and pH 9. With the same program the pH of an aqueous solution of 0.02 mol dm 3 Na2PCr is calculated to be 7.80 with PCr2 being present at a relative concentration of more than 99.9%. These results mean that any protonated or deprotonated form of PCr2 is present in negligible concentration and can, therefore, be ignored. The stability constants for NaPCr, KPCr and MgPCr were determined with the aid of the program HYPNMR-2000 by fitting all the data points (with unit weights) with the further inclusion of a stability constant for MgCl (log K /0.43) [5] as a constant entity. The refined stability constants are shown in Table 1. The Table 1 Logarithms of the formation constants of phosphocreatine complexes with sodium, potassium and magnesium ions at 37 8C, I 0.25 mol dm 3 set by addition of Me4NCl
Fig. 2. 31P chemical shifts of phosphocreatine at 37 8C as a function of added salt /phosphocreatine molar ratio. Hollow symbols represent the experimental points, solid lines refer to calculated titration curves. Chemical shifts were calculated on the basis of a constant analytical concentration of PCr equal to the average in each system: 0.01944 mol dm 3 for NaCl, and 0.2075 for KCl and MgCl2. (a) Experimental values and titration curves for the systems NaCl /PCr and KCl /PCr, ionic strength adjusted with Me4NCl; (b) experimental values and titration curve for the system MgCl2 /PCr, ionic strength adjusted with NaCl.
Reaction
Na PCr2 0 NaPCr K PCr2 0 KPCr Mg2 PCr2 0 MgPCr a
log K This work
Literature
0.590.2 0.390.2 1.4390.03
1.30 a 1.690.2
b
From [5]; T 25 8C, I 0.15 mol dm 3. From reference [4]; T 30 8C, in 0.1 mol dm 3 N -ethylmorpholine buffer at pH 8.0. b
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F. Cecconi et al. / Polyhedron 21 (2002) 1481 /1484
value obtained for the complex MgPCr is in satisfactory agreement with a previous determination [4], but with the complex KPCr our value is much smaller that the published value [5]. For NaPCr there is no previously reported value for comparison and the value is quite similar to that of the potassium complex, as expected. The chemical shifts of PCr2, NaPCr , KPCr and MgPCr were refined along with the stability constants. The values found are /2.2649/0.001, /0.99/0.6, /1.49/0.3 and /3.449/0.03 ppm, respectively. Agreement between observed and calculated resonances is excellent, as indicated by a value of 0.00085 ppm for the S.D. on all the residuals. The calculated resonances are also shown on Fig. 2. The good agreement also provides confirmation that the only complexes formed have 1:1 metal:PCr stoichiometry. Although the fit of the experimental data is very good the standard deviations calculated for both stability constants and chemical shifts of the sodium and potassium complexes are rather large. The main reason for this is that these complexes are rather weak so that even in the most concentrated solutions the degree of formation is small, 7.7% for NaCl /PCr and 8.7% for KCl/PCr. The magnesium complex is stronger and the degree of formation is much larger (reaching a maximum of 61%) so the standard deviations of both stability constant and chemical shift are an order of magnitude smaller. Two assumptions underlie the results given in this work. Firstly, that the magnetic susceptibility of the medium was not affected by the progressive replacement of Me4NCl by NaCl or KCl or of NaCl by MgCl2. Secondly, that the Me4N cation does not associate with PCr2. The validity of these assumptions was tested by replicating the NMR measurements in the series NaCl /PCr with two significant differences: Bu4PCl was added as an internal standard for the measurement of chemical shifts and Bu4NCl was used instead of Me4NCl for the background electrolyte. It is generally accepted that the tetrabutylammonium ion does not associate with phosphate-containing ligands [10,11]. The 31P chemical shift of the tetrabutylpho-
sphonium ion was found to be constant, 34.24569/ 0.0009 relative to external 85% H3PO4. This shows that variation of solution composition over the experimental range has no significant effect on bulk magnetic susceptibility. The second assumption was tested by comparing the calculated stability constants. With the tetrabutylammonium salt log K was found to be /0.49/ 0.3. This value is practically the same as the value obtained with the tetramethylammonium salt which demonstrates that neither cation interacts significantly with PCr.
Acknowledgements The authors thank MURST, Italy, for financial support of this work. Further support was received by the CNR target project Biotechnology, grant 97.01029.PF, and the ‘Fondazione Cassa di Risparmio di Bologna’.
References [1] I. Wadso¨, H. Gutfreund, P. Privlov, J.T. Edsall, W.P. Jenks, G.T. Strong, R.L. Biltonen, J. Biol. Chem. 251 (1976) 6879. [2] I. Wadso¨, R.L. Biltonen, Eur. J. Biochem. 153 (1985) 429. [3] R.M. Smith, A.E. Martell, NIST Critically Selected Stability Constants of Metal Complexes Database, US Department of Commerce, National Institute of Standards and Technology, Gaithersburg, MD, USA, 1997. [4] W.J. O’Sullivan, D.D. Perrin, Biochemistry 3 (1964) 18. [5] H.R. Halvorson, A.M.Q. Vande Linde, J.A. Helpern, K.M.A. Welch, NMR Biomed. 5 (1992) 53. [6] L.D. Pettit, H.K.J. Powell, IUPAC Stability Constants Database, Academic Software, Sourby Old Farm, Otley, LS21 2PW, UK, 1999. [7] C. Frassineti, S. Ghelli, P. Gans, A. Sabatini, M.S. Moruzzi, A. Vacca, Anal. Biochem. 231 (1995) 374. [8] G.W. Allen, P. Haake, J. Am. Chem. Soc. 98 (1976) 4990. [9] L. Alderighi, P. Gans, A. Ienco, D. Peters, A. Sabatini, A. Vacca, Coord. Chem. Rev. 184 (1999) 311. [10] R.M. Smith, R.A. Alberty, J. Phys. Chem. 60 (1956) 180. [11] R.M. Smith, R.A. Alberty, J. Am. Chem. Soc. 78 (1956) 2376.
Complex formation equilibria of phosphocreatine with sodium, potassium and magnesium ions Franco Cecconi a, Chiara Frassineti b, Peter Gans c, Stefano Iotti d, Stefano Midollini a, Antonio Sabatini e,*, Alberto Vacca e a I.S.S.E.C.C., Consiglio Nazionale delle Ricerche, I-50132 Firenze, Italy Dipartimento di Scienze Biomediche, Universita` di Modena, I-41100 Modena, Italy c School of Chemistry, University of Leeds, Leeds LS2 9JT, UK d Dipartimento di Medicina Clinica e Biotecnologia Applicata ‘D. Campanacci’, Universita` di Bologna, I-40138 Bologna, Italy e Dipartimento di Chimica, Universita` di Firenze, Via della Lastruccia, I-50019 Sesto Fiorentino, Italy b
Received 24 October 2001; accepted 15 November 2001
Abstract The formation of complexes between phosphocreatine, H2O3PNHC( /NH)N(CH3)CH2CO2H, and the ions Na , K and Mg2 have been investigated under physiological conditions (aqueous solution, T/37 8C and I /0.25 mol dm3) by means of 31P NMR spectroscopy. Only 1:1 complexes have been identified. Stability constants have been determined with the aid of the new computer program HYPNMR-2000. log10 K values were found to be/0.5(2),/0.3(2) and 1.43(3), respectively. The formation constant for the potassium complex is two orders of magnitude less that the literature value. # 2002 Published by Elsevier Science Ltd. Keywords: Phoshocreatine;
31
P NMR spectroscopy; Sodium complexes; Potassium complexes; Magnesium complexes
1. Introduction In the cells of tissues such as muscle and brain the high energy demand required for the cell function is satisfied by the ATP hydrolysis. The ATP is regenerated by the creatine kinase (CK) catalysed reaction (1) which is a magnesium-dependent transphosphorilation reaction. PCrADP 0 ATPCr
(1)
Cr stands for creatine, N -amidinosarcosine, and PCr for its N- phosphonic derivative, phosphocreatine.
* Corresponding author. Tel.: /39-055-475-3276; fax: /39-055354-845. E-mail address: [email protected] (A. Sabatini).
Each reagent in Eq. (1) should be understood [1,2] to include the various chemical species in the equilibrium system that contain it. For example, ATP is generally in equilibrium with its magnesium complex. The equilibrium constant of the reaction is usually reported in terms of the concentration of the free ionic forms. Since direct measurement of concentrations of the free ionic forms is not feasible, it is convenient to use an apparent ? , which is defined in terms of equilibrium constant, K CK ? the sums of the concentrations of all related forms. K CK is very useful operationally, but has the drawback of being dependent on both H and the concentrations of the principal metal ions which are present in the cytosolic medium, namely, Mg2, Na and K but not Ca2 whose influence can be neglected since, in vivo, it is almost completely bound to proteins such as calmodulin and calbinding D-proteins. To quantify this dependence the formation constants of all the species involved in the CK equilibrium with Mg2, Na and K need to be known. At physiological pH PCr is present as the anion PCr2 and the oxygen atoms on the phosphonate group can co-ordinate to the metal cations. Thus, PCr2 competes with other phosphate-
0277-5387/02/$ - see front matter # 2002 Published by Elsevier Science Ltd. PII: S 0 2 7 7 - 5 3 8 7 ( 0 2 ) 0 0 9 5 1 - 8
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containing species, such as HPO42, ATP and ADP, in forming metal-ion complexes. Few thermodynamic data have been reported for the formation of PCr complexes and those that have are so contradictory that it is stated that the published data do not meet the criteria required for a critical selection in the NIST database [3]. Formation constants have been published for MgPCr [4] and KPCr [5], but nothing has been reported for NaPCr . This is strange as the sodium and potassium complexes of ATP and ADP are usually quite similar [3,6]. Another disquieting aspect of the published data is the small difference between the formation constants of MgPCr and KPCr, since with analogous ligands magnesium complexes are much more stable than the corresponding potassium complexes [3,6]. Information regarding the stability of PCr complexes is essential for the determination of the concentrations of the species present in the cytosolic medium. We have, therefore, undertaken a systematic study, using 31P NMR spectroscopy, of complexation of the phosphate ligands PCr, ATP, ADP and AMP with the three cations Na , K e Mg2, under physiological conditions (T /37 8C and I /0.25 M). We now report the results for phosphocreatine.
2. Experimental 2.1. Chemicals Na2PCr ×/4.5H2O (Sigma) was used without further purification (Calc. for C4H17N3O9.5PNa2, C, 14.29; H, 5.10; N, 12.50. Found: C, 14.20; H, 5.21; N, 12.50%). The 31P NMR spectra of this substance show that hydrolysis is negligible in aqueous solution as the intensity of the phosphate ion signal was always less than 1% of the total integrated intensity. Analytical grade reagents (Merck) were used to prepare stock solutions of Me4NCl, NaCl, KCl and MgCl2 using weighed amounts of solute and solvent. D2O (99.8%, Merck) and reagent grade Bu4NCl (Fluka) and Bu4PCl (50% aqueous solution, Merck) were used as supplied. 2.2. NMR measurements 31
P{1H} NMR spectra were recorded at 81.02 MHz using a Bruker AC 200 spectrometer. The probe temperature was 37.09/0.2 8C. Field frequency stabilisation was established by using as solvent H2O /D2O (80:20 by volume). All chemical shifts are reported in ppm toward higher frequency relative to external 85% aqueous H3PO4. No corrections were made for differences in bulk susceptibly in the various samples. Digital resolution was 0.216 Hz per point. In all cases p/3 pulses were used. The feed was accumulated in 32 k data points
with zero filling to 64 k data points before Fourier transformation. The acquisition time was 4.62 s. 2.3. Spectra and compositions of the solutions used At least ten solutions were prepared for each system. Solutions were stored at approximately 4 8C to prevent hydrolysis. The concentration of Na2PCr was approximately 0.02 mol dm 3 in all solutions. The concentrations of the metal halides (NaCl, KCl and MgCl2) ranged between zero and the concentration corresponding to an ionic strength of 0.25 mol dm 3. Where necessary, the solutions were made upto constant ionic strength of 0.25 mol dm 3 by addition of Me4NCl for the NaCl /PCr and KCl /PCr systems or NaCl for the MgCl2 /PCr system. A further series of 11 replicate solutions in the NaCl /PCr system was prepared, but with Bu4PCl added as an internal reference and replacing Me4NCl by Bu4NCl. 2.4. Computational procedures In the course of this work the new computer program has been written and developed. This is a generalised program for the determination of stability constants from NMR chemical shift data in systems where exchange is rapid on the NMR time-scale. It has a novel structure. The main program is written in VISUAL BASIC and runs as a typical WINDOWS application. The main program contains extensive facilities for data input and preliminary calculations including the possibility of interactive data fitting with graphical displays of observed and calculated data. The stability constant refinement is a compiled FORTRAN module, called from the main program, which is based on the earlier HYPNMR program [7]. The refinement module reads data from an input file and records the results of the computation in an output file. On termination of the refinement the output file is automatically opened in the main program and is displayed in a customised viewer. HYPNMR-2000 can handle any number of individual resonances, including resonances from more than one nucleus, such as 13C and 1H, at the same time. There are no limitations on the number of reagents or stability constants that can be specified. Further details are available at http://www.chem.leeds.ac.uk/people/peter_gans/hypnmr.htm. HYPNMR-2000
3. Results and discussion All spectra showed a single narrow resonance, as illustrated in Fig. 1. This indicates that all the species related to phosphocreatine are in rapid equilibrium on the NMR time-scale. Confirmation of this state of affairs is provided by the smooth variation of the
F. Cecconi et al. / Polyhedron 21 (2002) 1481 /1484
Fig. 1. 31P NMR spectrum of a solution containing Na2PCr 0.020 mol dm 3; T/37 8C and I/0.25 mol dm 3, made up with Me4NCl.
chemical shift with changes in concentration of the metallic cations: with Na and K there is a slight drift to low field with increasing metal ion concentration and
1483
Fig. 3. Hyss distribution diagram of phosphocreatine as a function of pH. The pKa values were taken from Ref. [8].
with Mg2 there is a marked displacement to high field. These trends are illustrated in Fig. 2. Four protonation constants have been determined for phosphocreatine [8]. The three pKa values, /0.41, 2.84 and 4.46, were assigned to dissociation of one proton from the /PO3H2 group, dissociation of the carboxyl group proton and dissociation of a second proton from the phosphate group, respectively. The fourth pKa value, 11.2, relates to further deprotonation of PCr2. A diagram showing the distribution of the species as a function of pH is shown in Fig. 3. This diagram, produced by the program HYSS-2000 [9], shows that PCr2 is the only species present between pH 7 and pH 9. With the same program the pH of an aqueous solution of 0.02 mol dm 3 Na2PCr is calculated to be 7.80 with PCr2 being present at a relative concentration of more than 99.9%. These results mean that any protonated or deprotonated form of PCr2 is present in negligible concentration and can, therefore, be ignored. The stability constants for NaPCr, KPCr and MgPCr were determined with the aid of the program HYPNMR-2000 by fitting all the data points (with unit weights) with the further inclusion of a stability constant for MgCl (log K /0.43) [5] as a constant entity. The refined stability constants are shown in Table 1. The Table 1 Logarithms of the formation constants of phosphocreatine complexes with sodium, potassium and magnesium ions at 37 8C, I 0.25 mol dm 3 set by addition of Me4NCl
Fig. 2. 31P chemical shifts of phosphocreatine at 37 8C as a function of added salt /phosphocreatine molar ratio. Hollow symbols represent the experimental points, solid lines refer to calculated titration curves. Chemical shifts were calculated on the basis of a constant analytical concentration of PCr equal to the average in each system: 0.01944 mol dm 3 for NaCl, and 0.2075 for KCl and MgCl2. (a) Experimental values and titration curves for the systems NaCl /PCr and KCl /PCr, ionic strength adjusted with Me4NCl; (b) experimental values and titration curve for the system MgCl2 /PCr, ionic strength adjusted with NaCl.
Reaction
Na PCr2 0 NaPCr K PCr2 0 KPCr Mg2 PCr2 0 MgPCr a
log K This work
Literature
0.590.2 0.390.2 1.4390.03
1.30 a 1.690.2
b
From [5]; T 25 8C, I 0.15 mol dm 3. From reference [4]; T 30 8C, in 0.1 mol dm 3 N -ethylmorpholine buffer at pH 8.0. b
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F. Cecconi et al. / Polyhedron 21 (2002) 1481 /1484
value obtained for the complex MgPCr is in satisfactory agreement with a previous determination [4], but with the complex KPCr our value is much smaller that the published value [5]. For NaPCr there is no previously reported value for comparison and the value is quite similar to that of the potassium complex, as expected. The chemical shifts of PCr2, NaPCr , KPCr and MgPCr were refined along with the stability constants. The values found are /2.2649/0.001, /0.99/0.6, /1.49/0.3 and /3.449/0.03 ppm, respectively. Agreement between observed and calculated resonances is excellent, as indicated by a value of 0.00085 ppm for the S.D. on all the residuals. The calculated resonances are also shown on Fig. 2. The good agreement also provides confirmation that the only complexes formed have 1:1 metal:PCr stoichiometry. Although the fit of the experimental data is very good the standard deviations calculated for both stability constants and chemical shifts of the sodium and potassium complexes are rather large. The main reason for this is that these complexes are rather weak so that even in the most concentrated solutions the degree of formation is small, 7.7% for NaCl /PCr and 8.7% for KCl/PCr. The magnesium complex is stronger and the degree of formation is much larger (reaching a maximum of 61%) so the standard deviations of both stability constant and chemical shift are an order of magnitude smaller. Two assumptions underlie the results given in this work. Firstly, that the magnetic susceptibility of the medium was not affected by the progressive replacement of Me4NCl by NaCl or KCl or of NaCl by MgCl2. Secondly, that the Me4N cation does not associate with PCr2. The validity of these assumptions was tested by replicating the NMR measurements in the series NaCl /PCr with two significant differences: Bu4PCl was added as an internal standard for the measurement of chemical shifts and Bu4NCl was used instead of Me4NCl for the background electrolyte. It is generally accepted that the tetrabutylammonium ion does not associate with phosphate-containing ligands [10,11]. The 31P chemical shift of the tetrabutylpho-
sphonium ion was found to be constant, 34.24569/ 0.0009 relative to external 85% H3PO4. This shows that variation of solution composition over the experimental range has no significant effect on bulk magnetic susceptibility. The second assumption was tested by comparing the calculated stability constants. With the tetrabutylammonium salt log K was found to be /0.49/ 0.3. This value is practically the same as the value obtained with the tetramethylammonium salt which demonstrates that neither cation interacts significantly with PCr.
Acknowledgements The authors thank MURST, Italy, for financial support of this work. Further support was received by the CNR target project Biotechnology, grant 97.01029.PF, and the ‘Fondazione Cassa di Risparmio di Bologna’.
References [1] I. Wadso¨, H. Gutfreund, P. Privlov, J.T. Edsall, W.P. Jenks, G.T. Strong, R.L. Biltonen, J. Biol. Chem. 251 (1976) 6879. [2] I. Wadso¨, R.L. Biltonen, Eur. J. Biochem. 153 (1985) 429. [3] R.M. Smith, A.E. Martell, NIST Critically Selected Stability Constants of Metal Complexes Database, US Department of Commerce, National Institute of Standards and Technology, Gaithersburg, MD, USA, 1997. [4] W.J. O’Sullivan, D.D. Perrin, Biochemistry 3 (1964) 18. [5] H.R. Halvorson, A.M.Q. Vande Linde, J.A. Helpern, K.M.A. Welch, NMR Biomed. 5 (1992) 53. [6] L.D. Pettit, H.K.J. Powell, IUPAC Stability Constants Database, Academic Software, Sourby Old Farm, Otley, LS21 2PW, UK, 1999. [7] C. Frassineti, S. Ghelli, P. Gans, A. Sabatini, M.S. Moruzzi, A. Vacca, Anal. Biochem. 231 (1995) 374. [8] G.W. Allen, P. Haake, J. Am. Chem. Soc. 98 (1976) 4990. [9] L. Alderighi, P. Gans, A. Ienco, D. Peters, A. Sabatini, A. Vacca, Coord. Chem. Rev. 184 (1999) 311. [10] R.M. Smith, R.A. Alberty, J. Phys. Chem. 60 (1956) 180. [11] R.M. Smith, R.A. Alberty, J. Am. Chem. Soc. 78 (1956) 2376.