Complexation Studies on Inositol-Phosphates. 41, Alkali-Metal Complexes of Ddidyo-Inositol 1,2 ,Q Trisphosphate H. Bieth, G. Schlewer, and B. Spiess HIB,BS. Laboratoire
de Chimie Anaiytique, FaculG de Pharmacie de Strasbourg Illkirch, France. - GS. Centre de Neurochimie du CNR& DPpartement de Pharmacochimie Moi&ukaire, Strasbourg, France
ABSTIUCT properties of the D-myo-inosito! 1,2,6 trisphosphate (Ins(1,2,6)Ps) towards Li+, NaC, K+, Rb+, and Cs+ cations were studied at 25°C in a 0.1 M tetra-n-butylammonium bromide medium. For all cations, mononuclear and prownated species were found. For smaller cations (Li+, Na+, and K+) a dinuclear complex was also put into evidence. The main characteristic of the complexes is its high stability; and of the ligand, its nonselectivity. The Ins(1,2,6)P,-K system was ascertained using Sa;nrnartano’s methti which additionally enabled the influence of various K+ concentrations on the protonations constants to be considered.
The complexation
JNTRODUCTION Alkali-metal ions are qualitatively and_,quantitatively important in many biological systems, playing many vital roles, particularly in connection with membrane transport phenomena and nerve influx propagation. Interactions of these cations with xenobiotics and/or metabolic intermediates have been postulated or detected for a long time. Often such interactions are neglected in complexation studies due to their weakness. Nevertheless, such a simplification can sometimes lead to erroneous or incomplete speciation results which may not take into account species of low concentration but of major biological importance. The complexation ability in solution of natural and synthetic macrocyclic ligands towards alkali-metal cations have been, in general, the first to be investigated [l].
Address reprint requests to: Professor 3. Spiess, Laboratoire de Chimie Analytique, Pharmacie de Strasbourg, 74 Route du Rhin, 67401 Illkirch, France. Journal of Inorganic Biochemistry, 41, 37-45 (1Wl) 0 1991 Elsevier Science Publishing Co., Inc.. 655 Avenue of the Americas,
FacultC de 37
NY, NY 10010
0162-0134/91/$3.50
38
W. Bieth et al.
More recently, Sammartano et al. have focused their attention on the weak interactions of alkali ions and a wide variety of ligands including carboxylates, amines, amino acids, sulphates, and phosphates [2-61. Among the latter, pyrophosphates (PP), tripolyphosphates (TPP) [2], and ATP [3] have been particularly considered. Due to the problems that ouch studies pose, the authors also proposed a methodological approach for the determination of weak complex formation constants by potentiometric techniques 173. Previous studies on the protonation [S, 91 and the complexation properties [lo] of inositol-phosphates performed in our laboratory demonstrated that the presence of alkali cations greatly influences these properties by competing with the cations under investigation. For example, the protonation constants of phytic acid, the hexaphosphorylated ester of myo-inositol, are largely dependent on the concentration of the supporting electrolyte if it contains sodium or potassium cations [8]. Even for less phosphorylated esters such as myo-inositol trisphosphates, the effect of these cations remains important on both the nature and the stability of the species in solution [lo]. Since much attention has been paid in the last few years to certain members of the family of the inositol-phosphates due to their second messenger properties [1 1- 193 or other biological actions [20, 211, it seems worthwhile to consider the interactions of such ligands with alkali-metal cations. Indeed, whatever the biological effect these ligands may have, it is the result of interactions at a molecular level which can be strongly influenced by the ionic environment [22]. As alkali-metals are present in various compartments of the living organisms and sometimes undergo fast local concentration variations at the cellular level, they are likely to be involved in any genera! imechanism of action of the inositol-phosphates. In this work the complexation properties of the D-myo-inositol 1,2,6 trisphosphate’ (Ins(l,2,6)P,) towards Li+, Na+, K+, Rb+, and Cs+ were studied after having previously considered its calcium and magnesium complexes. For all the five cations the study was performed at 25°C in a 0.1 M tetra-n-butylammonium bromide (But,NBr) medium and the data were interpreted by the program MINIQUAD [23]. In addition, for potassium, the method of Sammartano et al. was used along with their program ES2WC 1 1241.
EXPERIMENTAL Materials Hydrated Na,HIns( 1,2,6)P,, provided by PERSTORP PHARbIA (Sweden) was converted into its acidic form as previously described [8- lo]. Reagent grade sodium, potassium, and rubidium chlorides and Suprapure lithium and cesium bromides were used. The base used as titration reactant was a solution of tetramethylammonium hydroxide prepared in carbonate-free water. The concentration of the ligand was determined from the potentiometric titration curve, and the concentrations of the metal salts obtained either by emission spectroscopy (Li+, Na+, K+, Rb+) or by weight (Cs+). All the precautions taken in the experiments were the same as those indicated for the previous studies [8-lo].
’ Another nomenclature trisphosphate
[30, 311.
often used in organic
chemistry
is for this compound:
sn-mnyo-inositol
1,2,6
COMPLEXATION
Potentiometric
STUDIES
ON INOSITOL-PHOSPHATES
39
Measurements
Method of CompeZition with the Proton. The pff (pU means the cologarithm of the concentration of the proton) measurements made during the titration of ligand and metal mixtures with a base contain the information related to the nature and stability of the complexes in solution, provided that the protonation constants and the analytical concentrations of the reactants are known. The experimental protocol and the apparatus were the same as these employed for studying the calcium and magnesium complexes. Only the metal-to-ligand ratios are different and in general much higher (from 2 to 10) due to the tower stability of the complexes and to fewer problems with hydrolysis and precipitation. As a consequence the ii vs pN curves (Ft being the number of ligands bound per mole of metal) gave almost no information about the nature and the stability of the species. Thus, the constants pXyz referring to the following general equilibria: xM++ were obtained
by computing
yH++
zL6-*
the experimental
MXH,L’,6”-x-y1data with the program
MINIQUAD.
Sammartano et al. ‘s Method and Use of ES2 WCI [24]. ln order to check the resuhs determined by the previous program, the method proposed by Sammartano et ai. was carried out with potassium. The program ES2WCl calculates the constants oXyz by minimizing the function:
where j5 and p’ are, respectively, the calculated and experimental mean number of protons bound per mole of ligand in media of various ionic strengths which were kept constant by the cation under study. ‘p’ is obtained from the conditional protonation constants &, corresponding to the equilibria:
L6-+
yH+aHyL’b-Y)-,
which take place in six different ionic backgrounds (KCl, 0.01; 0.06; 0. IO; 0.16; 0.50; 1.00 mol.dm-“). The measurements were carried out with an Ingold combined electrode connected to an Isis 20,000 pH meter. Data acquisition and the control of the buret were performed by the program MICROPOT [25] running on an Apple II computer. The calibration of the pH glass eIectrode was made in each medium by titration of hydrochloric acid (4.0 mmol.dmB3) with tetramethylammonium hydroxide (Me,NOH, 2.0 mmol.dm’3). The emf measurements and the titrant volumes analyzed by SUPERQUAD gave a set of 6 E, and pK, values. The titrations were carried out at 25°C f 0.1 “C under an inert atmosphere on 10 cm3 of solutions containing the ligand (1.7 mmol.dm-3) in its acidic form, and various amounts of potassium ions (0.01-1.00 mol.dm-3). The titration reactant was a solution of Me,NOH (0.040 mol.dmh3) having the same concentration of K4 as the medium under study. The conditional constants in the various backgrounds were obtained by treating the data with the program GRAPHIPOT [9]. In order to compare the conditional constants to the protonation constants obtained in the absence of the
40
E?. Bieth et af.
TABLE 1. Logarithm of the Overall Stability Constants of the Complexes
Formed by Alkali-Metals with Ins(S ,2,6)P,. The Stoichiometry of the Complexes is Given According to the General Formula Indicated in the Text. S and R Correspond Respectively to the Standard Deviation of the Residuals and the R Factor Given by MINIQUAD. n is the To*4 Number of Observations Used for the Calculations Obtained in N experiments.
Cation
x
Y
2
H+
0 0 0 0
1 2 3 4
1 1 1 1
9.48 f 0.01 16.70 f 0.01 22.40 f 0.01 24.80 f 0.01
Li+
1 1 1 2
0 1 2 0
1 1 1 1
2.38 11.24 17.83 3.60
f f f *
1 1 1 2
0 1 2 0
1 1
2.32 11.29 17.93 3.49
f f f f
1
0
1
1
1
1
1
2
1
2
0
I
2.58 2.58 11.46f Il.18 18.06 l’T.38 3.84 3.09
Rb+
1 1 1
0 1 2
1 1
Gi+
1 1 1 2 2
0 1 2 0 1
1 1 1 1 1
Na+
K+
interfering
cation,
1 1
1
S
R
2.2-9.9
369
6.70 1O-6
O.OOl6
0.0.02 0.02 0.04 0.07
3.6-9.4
366
1.08 10-S
0.0012
0.02 0.01 0.03 0.05
3.1-9.6
248
6.78 IO-6
0.0007
f 0.01 f 0.05 0.01 + 0.03 f 0.02 f 0.05 f 0.03 f 0.09
3.3-9.7
398
7.25 IG”
O.OMI8
2.57 f 0.01 11.32 f 0.02 17.80 f 0.04
3.3-9.7
359
1.31 10”
Ci.001
3.3-9.7
361
9.46 10’”
0.0010
performed,
in which
2.51 11.32 17.87 (2.94 (12.19
f f f f f
0.01 0.03 0.03 0.33) 0.18)
the same experiments
the KC1 was replaced RESULTS
n
log B,,, f 0
pH Range
described
N
above
were
by But,NBr.
AND DISCUSSION The nature and stability of the complexes that form Ins(1,2,6)& with IA’, Nae, Ke, Rb+. and Cs+ are reported in Table 1. These constants are the result of a final refmement of n points corresponding to N experiments. Individual refinements could lead, for some curves, to a somewhat different model due to the various metal-toligand ratios used. The results show in all cases the existence of three mononuclear complexes: an ML complex accompanied with a monoprotonated and diprotonated species. In addition, for the smaller cations, Li+, Na+, and K+, a dinuclear complex is well defined, whereas for the larger cations, Rb+ and Cs+, the same species either did not converge (Rb+), or converged with a low value and a bad confidence interval (cs+).
COMPLEXATION
STUDIES
41
ON INOSITOL-PHOSPHATES
TABLE 2. Logarithm of the Stability Constants of Alkali-Metal Complexes with some Polycarboxylates and Phosphates. (a) Ref. 3. (5) Ref. 26, (c) Ref. 5
EDTA NTA PP TPP ATP
Li+
Na+
2.9v 2.56’
1.848 1.358 1.33= 1 .43p 1.32’
1.79=
K+ 0.80’ 0.79= 1.51’ 1SF 1.21C
Rb+
CS’
0.59b
O.lJb
1.24’
1.20=
Concerning the stability of the complexes, it appears that they are quite high for alkali-metals and that the complexes do not display any selectivity of complexation. The mean value of the ML stability constants is 2.47 f 0.12. This value is much higher than those encountered for polycarboxylates, including NTA and EDTA (except for the Li-NTA and Li-EDTA complexes), and also for ATP and polyphosphates. For co,mparison, some of these values are indicated in ‘Table 2. The second characteristics of the Ins(l,2,6)P, ligand is its nonselectivity towards the alkali-cations. For all the mononuclear species, there is, from Li+ to Cs+, no incremental or decremental trend and no maximum of complexation. Such behavior differs markedly from that observed with polycarboxylates where the stability of the lithium complexes is much higher than those of the larger cations. Carboxylates favor small cations over large ones. On the other hand, as can be seen in Table 2, for phosphates the variations of stability constants with the size of the cation are much lower. For ATP, the cesium, rubidium, and potassium complexes behave similarly, and the sodium complex is only slightly more stable. It can also be observed that the Na-diphosphate and Na-tripolyphosphate complex,es are, like the Na-Ins( 1,2,6)P, complex, a little less stable than the potassium complexes. These results may partly be explained by the structure of the ligand which exhibits its three phosphate groups around the inositol moiety like three arms. In each possible ring conformation, these arms are able to move freely and multidirectionally below and above the mean plane of the ring (see the ligand in one I-ax/2-eq conformation given by ALCHEMY’). Such an “octopus-like” ligand may then easily adapt to the roughly spherical shape of the cations even though the size of these spheres may vary greatly from Li+ to Cs’. In addition, for phosphorylated sugars as shown in the solid state, there are many possible ligand oxygen atoms and no general preference for any particular kind of oxygen is found. For example, sodium does not bind the phosphate oxygen atoms in sodium inosine 5’-monophosphate hexalhydrate [27] whereas the potassium cation does have such bonds in the glucose-l-phosphate salts [28]. The Ins(1,2,6)P, also displays many coordination sites which will not be necessarily the same for all the cations. The mean stepwise stability constants KK, and KE, , respectively related to the equilibria M++ HL’-I=s MHL4- and M++ H,L4’* MH,L3-, are equal to 1.84 f 0.08 and 1.20 f 0. IO. These constants show, for the calcium and magnesium
2 TRIP0S Associates, St.
Louis,
Missouri.
42
H. Bieth et al.
SCHEME
1.
complexes [lo], that the mono and diprotonated species still have an appreciable stability. Besides the mononuclear complexes, homodinuclear complexes have undoubtedly been found for the three smaller cations, though their existence is uncertain for Cs’ and absent for Rb+‘. Such complexes were already described for PP and TPP with Na+ and IS+, for ATP with Li +, by Sammartano et al. 13, 5) and with Lif, Na+‘, and M+ by Botts et al. [29]. In the case of Ins(l,2,6)P, it seems that the formation of dinuclear complexes is hindered by the size of the larger cations. The distribution curves of the complexes which form in conditions where the metal to ligand concentration is equal to 10 are illustrated in Figure 1. It appears that, over the whole pN range considered, the two main potassium complexes are ML and MHL whereas MH 2 L and M, L remain under by about 15%. In these concentration conditions, at a physiological pH, 33% and 6% of the Ins(l,2,6)P, are engaged, respectively, in the mono and diprotonated complexes. This shows that, in biological media where the metal-to-ligand ratios may presumably reach values of 10-100, it is impossible to neglect the complexed forms of the ligand. It can be expected that other inositol-trisphosphates, among them those playing an intracellular biological action, would behave similarly. Close to the cell membrane,
M.H+/C:
1. Distribution curves of the K-H-Ins( 1,2,6)P, species plotted against
,100
.*_....*...‘-. : HA ._
FIGURE
.
---
_ .
m. . -.-.. * -.
PH
ML HL
pH. Co, = 0.001 mol.dmB3, Co, = 0.010 mol.dmW3.
COMPLEXATION
TABLE
STUDIES
43
ON INOSITOL-PHOSPHATES
3. Logarithm
of the Stepwise Conditional Protonation Constants of the Ins( 1,2,6)P,-H System in Two Different Media and Ionic Strength Conditions KC1
Cont. mo! - dm-’
But4 NBr
WsK,
logKz
0.06 0.10
9.13 8.66 8.56
7.22 6.77 6.71
5.82 5.53 5.47
0. I6 0.50 1.00
8.41 8.15 8.02
6.57 6.27 6.15
5.34 5.16 5.02
0.01
bK3
QK,
bK2
loisK3
9.52 9.44 9.45 (9.48) 9.34 9.36 9.32
7.33 7.19 7.21 (7.22) 7.11 7.16 7.17
5.86 5.75 5.73 (5.70) 5.68 5.69 5.80
by opening
ionic channels, the local concentration of cations may vary in !arge proportions. As these inositol-phosphates bind membrane receptors, it is likely that the cations may influence the binding to the receptor and by doing so play a role in the control of the signal. The results obtained by Sammartano’s method for K+ are given in Table I in italics. It can be seen that even though the method of cletermination is completely different, the values of the constants for the ML and MHL complexes are in line with those obtained by the method of competition with the proton. For the MH, L and M,L complexes, the values are lower, but it should be noted that both these species correspond to minor species in regard to the previous ones (see Fig. 1). protonation constants, are reported in The values of log K,, stepwise conditional Table 3 and Figure 2. The values determined in the same medium by MLNIQUAD are given in parenthesis. The agreement between both sets of values is good. The comparison between curves (a) and (b), corresponding to the But,NBr and KC1 media shows the strong competition between the potassium and the proton. The effect is especially high for the protonation step 1 and 2, i.e., pH of 6-9, and for supporting-eIectrolyte concentrations ranging from O-O.2 M. Both these conditions are encountered in biological media. It still appears that the influence of alkali-cations must be taken into consideration in both biological and physico-chemical reactions involving inositol-phosphates.
0
FIGURE 2. Curves of the variation of the stepwise protonation constants vs the concentrations of supporting-electrolyte. (a) But,NBr Media; rb) KC1 Media.
-_
l -? . . . .. . supporllnp-tlcctmlyL
(II)
44
Ei. Bieth
et al.
The authors are grateful to PERSTORP PHARMA (Sweden) for providing Ins(l.t,6)P,, and thank Miss Caroline Dietrich for her technical assistance.
the
REFERENCES 1. J. M. Lehn, in Structure and bonding, J. D. Dunitz, Ed., Springer-Verlag. New York, 1973. Anal. Chem. 57, 2956 (1985). 2. P. G. Daniele, C. Rigano, and S. Sammartano, 3. P. G. Daniele, C. Rigano, and S. Sammartano, Talanta 30, 81 (1985). 4. S. Capone, A. De Robertis, C. De Stefano, S. Sammartano, R. Scarcella, and C. Rigano. Thermochim. Acra 86, 273 (1985). 5. A. De Robertis, C. De Stefano, S. Sammartano, R. Cali, R. Purrello, R. Scarcella, C. Rigano, J. Chem. Research (S). 164 (1986); J. Chem. Research (M), 1301 (1986). 6. A. Casale, A. De Robenis, C. De Steiano, Thermochim. Acta 128. 261 (1988). Talanta 34(13), 233 (1987). 7. A. De Robertis, C. De Stefano, S. Sammartano, 8. H. Bieth and B. Spiess, J. Chem. Sot., Faraday Trans. I 82, :935 (1986). 9. H. Bieth, P. Jost, B. Spiess, and C. Wehrer. Anal. Lett. 22(3), 703 (1989). 10. H. Bietb, P. Jost, B. Spiess, J. Inorg. Biochern., in press (1989). 11. M. J. Bet-ridge and R. F. Irvine, Nuture 306, 67 (1984). 12. M. 3. Berridge and R. F. Irvine, Nature 312, 315 (1984). 13. M. J. Bet-ridge, Biochem. J. 220, 345 (1984). 14. Ata A. Abdel-Latif, PhQrmaco~ogica~ Reviews 38(3), 227 (1986). 15. R. Parthasarathy and F. Eisenberg, Biochem. J. 235, 313 (1986). 16. J. W. Putney, D. L. Aub, C. W. Taylor, and J. E. Merritt, Fed. Proc. 45, 2634 (1986). 17. B. W. Agranoff, Fed. Proc. 45, 2629 (1986). 18. D. Lichtstein and D. Rodbard, Ltye Sciences 40, 2041 (1987). 19. M. J. Berridge and R. F. Irvine, Nature 341, 197 (1989). 20. M. J. Vallejo, T. Jackson, S. Lightman, and M. R. Hanleyt, Nature 330, 656 (1987). composition and a method for preparing inositoltrisphos21. Siren Matti, A pharmaceutical phate, U.S. Patent No. 4735936. 22. P. F. Worley, J. M. Baraban. S. Supattapone, V. S. Wilson, and S. H. Snyder, J. Biol. Chem. 262, 12132 (1987). 23. A. Sabatini, A. Vacca, and P. Gans, Tafunta 21, 53 (1974). 24. A. De Robertis, C. De Stefano, S. Sammartano, and C. Rigano. Talanta 34(11). 933 (1987). 25. P. Jost and B. Heulin, L’actualite chimique, Avril, 34 (1984). 26. L. 0. Sillen. A. E. Mat-tell, Stability constants of metual-ion complexes, Special publications No. 17 (1964) and No. 25 (1971), ChemicaI Society, London. 27. S. T. Rao and J. Sundaralingam, J. Am. Chem. Sot. 91, 1210 (1969). 28. C. A. Beevers and G. H. Maconochie, Acta Cryst. 18, 232 (1965). 29. J. Botts, A. Chashin, and L. Young, Biochemistry 4, 1788 (1965). 30. B. A. Klyashchitskii, V. 1. Shvets, and W. A. Preobrazhensky. Chem. Phys. Lipids 3, 393 (1969). 31. V. I. Shvets and R. P. Evstigneeva, Zhur. Org. Khemi 8, 1550 (1972).
Received April 4, 1990; accepted April X9, 1990
de Chimie Anaiytique, FaculG de Pharmacie de Strasbourg Illkirch, France. - GS. Centre de Neurochimie du CNR& DPpartement de Pharmacochimie Moi&ukaire, Strasbourg, France
ABSTIUCT properties of the D-myo-inosito! 1,2,6 trisphosphate (Ins(1,2,6)Ps) towards Li+, NaC, K+, Rb+, and Cs+ cations were studied at 25°C in a 0.1 M tetra-n-butylammonium bromide medium. For all cations, mononuclear and prownated species were found. For smaller cations (Li+, Na+, and K+) a dinuclear complex was also put into evidence. The main characteristic of the complexes is its high stability; and of the ligand, its nonselectivity. The Ins(1,2,6)P,-K system was ascertained using Sa;nrnartano’s methti which additionally enabled the influence of various K+ concentrations on the protonations constants to be considered.
The complexation
JNTRODUCTION Alkali-metal ions are qualitatively and_,quantitatively important in many biological systems, playing many vital roles, particularly in connection with membrane transport phenomena and nerve influx propagation. Interactions of these cations with xenobiotics and/or metabolic intermediates have been postulated or detected for a long time. Often such interactions are neglected in complexation studies due to their weakness. Nevertheless, such a simplification can sometimes lead to erroneous or incomplete speciation results which may not take into account species of low concentration but of major biological importance. The complexation ability in solution of natural and synthetic macrocyclic ligands towards alkali-metal cations have been, in general, the first to be investigated [l].
Address reprint requests to: Professor 3. Spiess, Laboratoire de Chimie Analytique, Pharmacie de Strasbourg, 74 Route du Rhin, 67401 Illkirch, France. Journal of Inorganic Biochemistry, 41, 37-45 (1Wl) 0 1991 Elsevier Science Publishing Co., Inc.. 655 Avenue of the Americas,
FacultC de 37
NY, NY 10010
0162-0134/91/$3.50
38
W. Bieth et al.
More recently, Sammartano et al. have focused their attention on the weak interactions of alkali ions and a wide variety of ligands including carboxylates, amines, amino acids, sulphates, and phosphates [2-61. Among the latter, pyrophosphates (PP), tripolyphosphates (TPP) [2], and ATP [3] have been particularly considered. Due to the problems that ouch studies pose, the authors also proposed a methodological approach for the determination of weak complex formation constants by potentiometric techniques 173. Previous studies on the protonation [S, 91 and the complexation properties [lo] of inositol-phosphates performed in our laboratory demonstrated that the presence of alkali cations greatly influences these properties by competing with the cations under investigation. For example, the protonation constants of phytic acid, the hexaphosphorylated ester of myo-inositol, are largely dependent on the concentration of the supporting electrolyte if it contains sodium or potassium cations [8]. Even for less phosphorylated esters such as myo-inositol trisphosphates, the effect of these cations remains important on both the nature and the stability of the species in solution [lo]. Since much attention has been paid in the last few years to certain members of the family of the inositol-phosphates due to their second messenger properties [1 1- 193 or other biological actions [20, 211, it seems worthwhile to consider the interactions of such ligands with alkali-metal cations. Indeed, whatever the biological effect these ligands may have, it is the result of interactions at a molecular level which can be strongly influenced by the ionic environment [22]. As alkali-metals are present in various compartments of the living organisms and sometimes undergo fast local concentration variations at the cellular level, they are likely to be involved in any genera! imechanism of action of the inositol-phosphates. In this work the complexation properties of the D-myo-inositol 1,2,6 trisphosphate’ (Ins(l,2,6)P,) towards Li+, Na+, K+, Rb+, and Cs+ were studied after having previously considered its calcium and magnesium complexes. For all the five cations the study was performed at 25°C in a 0.1 M tetra-n-butylammonium bromide (But,NBr) medium and the data were interpreted by the program MINIQUAD [23]. In addition, for potassium, the method of Sammartano et al. was used along with their program ES2WC 1 1241.
EXPERIMENTAL Materials Hydrated Na,HIns( 1,2,6)P,, provided by PERSTORP PHARbIA (Sweden) was converted into its acidic form as previously described [8- lo]. Reagent grade sodium, potassium, and rubidium chlorides and Suprapure lithium and cesium bromides were used. The base used as titration reactant was a solution of tetramethylammonium hydroxide prepared in carbonate-free water. The concentration of the ligand was determined from the potentiometric titration curve, and the concentrations of the metal salts obtained either by emission spectroscopy (Li+, Na+, K+, Rb+) or by weight (Cs+). All the precautions taken in the experiments were the same as those indicated for the previous studies [8-lo].
’ Another nomenclature trisphosphate
[30, 311.
often used in organic
chemistry
is for this compound:
sn-mnyo-inositol
1,2,6
COMPLEXATION
Potentiometric
STUDIES
ON INOSITOL-PHOSPHATES
39
Measurements
Method of CompeZition with the Proton. The pff (pU means the cologarithm of the concentration of the proton) measurements made during the titration of ligand and metal mixtures with a base contain the information related to the nature and stability of the complexes in solution, provided that the protonation constants and the analytical concentrations of the reactants are known. The experimental protocol and the apparatus were the same as these employed for studying the calcium and magnesium complexes. Only the metal-to-ligand ratios are different and in general much higher (from 2 to 10) due to the tower stability of the complexes and to fewer problems with hydrolysis and precipitation. As a consequence the ii vs pN curves (Ft being the number of ligands bound per mole of metal) gave almost no information about the nature and the stability of the species. Thus, the constants pXyz referring to the following general equilibria: xM++ were obtained
by computing
yH++
zL6-*
the experimental
MXH,L’,6”-x-y1data with the program
MINIQUAD.
Sammartano et al. ‘s Method and Use of ES2 WCI [24]. ln order to check the resuhs determined by the previous program, the method proposed by Sammartano et ai. was carried out with potassium. The program ES2WCl calculates the constants oXyz by minimizing the function:
where j5 and p’ are, respectively, the calculated and experimental mean number of protons bound per mole of ligand in media of various ionic strengths which were kept constant by the cation under study. ‘p’ is obtained from the conditional protonation constants &, corresponding to the equilibria:
L6-+
yH+aHyL’b-Y)-,
which take place in six different ionic backgrounds (KCl, 0.01; 0.06; 0. IO; 0.16; 0.50; 1.00 mol.dm-“). The measurements were carried out with an Ingold combined electrode connected to an Isis 20,000 pH meter. Data acquisition and the control of the buret were performed by the program MICROPOT [25] running on an Apple II computer. The calibration of the pH glass eIectrode was made in each medium by titration of hydrochloric acid (4.0 mmol.dmB3) with tetramethylammonium hydroxide (Me,NOH, 2.0 mmol.dm’3). The emf measurements and the titrant volumes analyzed by SUPERQUAD gave a set of 6 E, and pK, values. The titrations were carried out at 25°C f 0.1 “C under an inert atmosphere on 10 cm3 of solutions containing the ligand (1.7 mmol.dm-3) in its acidic form, and various amounts of potassium ions (0.01-1.00 mol.dm-3). The titration reactant was a solution of Me,NOH (0.040 mol.dmh3) having the same concentration of K4 as the medium under study. The conditional constants in the various backgrounds were obtained by treating the data with the program GRAPHIPOT [9]. In order to compare the conditional constants to the protonation constants obtained in the absence of the
40
E?. Bieth et af.
TABLE 1. Logarithm of the Overall Stability Constants of the Complexes
Formed by Alkali-Metals with Ins(S ,2,6)P,. The Stoichiometry of the Complexes is Given According to the General Formula Indicated in the Text. S and R Correspond Respectively to the Standard Deviation of the Residuals and the R Factor Given by MINIQUAD. n is the To*4 Number of Observations Used for the Calculations Obtained in N experiments.
Cation
x
Y
2
H+
0 0 0 0
1 2 3 4
1 1 1 1
9.48 f 0.01 16.70 f 0.01 22.40 f 0.01 24.80 f 0.01
Li+
1 1 1 2
0 1 2 0
1 1 1 1
2.38 11.24 17.83 3.60
f f f *
1 1 1 2
0 1 2 0
1 1
2.32 11.29 17.93 3.49
f f f f
1
0
1
1
1
1
1
2
1
2
0
I
2.58 2.58 11.46f Il.18 18.06 l’T.38 3.84 3.09
Rb+
1 1 1
0 1 2
1 1
Gi+
1 1 1 2 2
0 1 2 0 1
1 1 1 1 1
Na+
K+
interfering
cation,
1 1
1
S
R
2.2-9.9
369
6.70 1O-6
O.OOl6
0.0.02 0.02 0.04 0.07
3.6-9.4
366
1.08 10-S
0.0012
0.02 0.01 0.03 0.05
3.1-9.6
248
6.78 IO-6
0.0007
f 0.01 f 0.05 0.01 + 0.03 f 0.02 f 0.05 f 0.03 f 0.09
3.3-9.7
398
7.25 IG”
O.OMI8
2.57 f 0.01 11.32 f 0.02 17.80 f 0.04
3.3-9.7
359
1.31 10”
Ci.001
3.3-9.7
361
9.46 10’”
0.0010
performed,
in which
2.51 11.32 17.87 (2.94 (12.19
f f f f f
0.01 0.03 0.03 0.33) 0.18)
the same experiments
the KC1 was replaced RESULTS
n
log B,,, f 0
pH Range
described
N
above
were
by But,NBr.
AND DISCUSSION The nature and stability of the complexes that form Ins(1,2,6)& with IA’, Nae, Ke, Rb+. and Cs+ are reported in Table 1. These constants are the result of a final refmement of n points corresponding to N experiments. Individual refinements could lead, for some curves, to a somewhat different model due to the various metal-toligand ratios used. The results show in all cases the existence of three mononuclear complexes: an ML complex accompanied with a monoprotonated and diprotonated species. In addition, for the smaller cations, Li+, Na+, and K+, a dinuclear complex is well defined, whereas for the larger cations, Rb+ and Cs+, the same species either did not converge (Rb+), or converged with a low value and a bad confidence interval (cs+).
COMPLEXATION
STUDIES
41
ON INOSITOL-PHOSPHATES
TABLE 2. Logarithm of the Stability Constants of Alkali-Metal Complexes with some Polycarboxylates and Phosphates. (a) Ref. 3. (5) Ref. 26, (c) Ref. 5
EDTA NTA PP TPP ATP
Li+
Na+
2.9v 2.56’
1.848 1.358 1.33= 1 .43p 1.32’
1.79=
K+ 0.80’ 0.79= 1.51’ 1SF 1.21C
Rb+
CS’
0.59b
O.lJb
1.24’
1.20=
Concerning the stability of the complexes, it appears that they are quite high for alkali-metals and that the complexes do not display any selectivity of complexation. The mean value of the ML stability constants is 2.47 f 0.12. This value is much higher than those encountered for polycarboxylates, including NTA and EDTA (except for the Li-NTA and Li-EDTA complexes), and also for ATP and polyphosphates. For co,mparison, some of these values are indicated in ‘Table 2. The second characteristics of the Ins(l,2,6)P, ligand is its nonselectivity towards the alkali-cations. For all the mononuclear species, there is, from Li+ to Cs+, no incremental or decremental trend and no maximum of complexation. Such behavior differs markedly from that observed with polycarboxylates where the stability of the lithium complexes is much higher than those of the larger cations. Carboxylates favor small cations over large ones. On the other hand, as can be seen in Table 2, for phosphates the variations of stability constants with the size of the cation are much lower. For ATP, the cesium, rubidium, and potassium complexes behave similarly, and the sodium complex is only slightly more stable. It can also be observed that the Na-diphosphate and Na-tripolyphosphate complex,es are, like the Na-Ins( 1,2,6)P, complex, a little less stable than the potassium complexes. These results may partly be explained by the structure of the ligand which exhibits its three phosphate groups around the inositol moiety like three arms. In each possible ring conformation, these arms are able to move freely and multidirectionally below and above the mean plane of the ring (see the ligand in one I-ax/2-eq conformation given by ALCHEMY’). Such an “octopus-like” ligand may then easily adapt to the roughly spherical shape of the cations even though the size of these spheres may vary greatly from Li+ to Cs’. In addition, for phosphorylated sugars as shown in the solid state, there are many possible ligand oxygen atoms and no general preference for any particular kind of oxygen is found. For example, sodium does not bind the phosphate oxygen atoms in sodium inosine 5’-monophosphate hexalhydrate [27] whereas the potassium cation does have such bonds in the glucose-l-phosphate salts [28]. The Ins(1,2,6)P, also displays many coordination sites which will not be necessarily the same for all the cations. The mean stepwise stability constants KK, and KE, , respectively related to the equilibria M++ HL’-I=s MHL4- and M++ H,L4’* MH,L3-, are equal to 1.84 f 0.08 and 1.20 f 0. IO. These constants show, for the calcium and magnesium
2 TRIP0S Associates, St.
Louis,
Missouri.
42
H. Bieth et al.
SCHEME
1.
complexes [lo], that the mono and diprotonated species still have an appreciable stability. Besides the mononuclear complexes, homodinuclear complexes have undoubtedly been found for the three smaller cations, though their existence is uncertain for Cs’ and absent for Rb+‘. Such complexes were already described for PP and TPP with Na+ and IS+, for ATP with Li +, by Sammartano et al. 13, 5) and with Lif, Na+‘, and M+ by Botts et al. [29]. In the case of Ins(l,2,6)P, it seems that the formation of dinuclear complexes is hindered by the size of the larger cations. The distribution curves of the complexes which form in conditions where the metal to ligand concentration is equal to 10 are illustrated in Figure 1. It appears that, over the whole pN range considered, the two main potassium complexes are ML and MHL whereas MH 2 L and M, L remain under by about 15%. In these concentration conditions, at a physiological pH, 33% and 6% of the Ins(l,2,6)P, are engaged, respectively, in the mono and diprotonated complexes. This shows that, in biological media where the metal-to-ligand ratios may presumably reach values of 10-100, it is impossible to neglect the complexed forms of the ligand. It can be expected that other inositol-trisphosphates, among them those playing an intracellular biological action, would behave similarly. Close to the cell membrane,
M.H+/C:
1. Distribution curves of the K-H-Ins( 1,2,6)P, species plotted against
,100
.*_....*...‘-. : HA ._
FIGURE
.
---
_ .
m. . -.-.. * -.
PH
ML HL
pH. Co, = 0.001 mol.dmB3, Co, = 0.010 mol.dmW3.
COMPLEXATION
TABLE
STUDIES
43
ON INOSITOL-PHOSPHATES
3. Logarithm
of the Stepwise Conditional Protonation Constants of the Ins( 1,2,6)P,-H System in Two Different Media and Ionic Strength Conditions KC1
Cont. mo! - dm-’
But4 NBr
WsK,
logKz
0.06 0.10
9.13 8.66 8.56
7.22 6.77 6.71
5.82 5.53 5.47
0. I6 0.50 1.00
8.41 8.15 8.02
6.57 6.27 6.15
5.34 5.16 5.02
0.01
bK3
QK,
bK2
loisK3
9.52 9.44 9.45 (9.48) 9.34 9.36 9.32
7.33 7.19 7.21 (7.22) 7.11 7.16 7.17
5.86 5.75 5.73 (5.70) 5.68 5.69 5.80
by opening
ionic channels, the local concentration of cations may vary in !arge proportions. As these inositol-phosphates bind membrane receptors, it is likely that the cations may influence the binding to the receptor and by doing so play a role in the control of the signal. The results obtained by Sammartano’s method for K+ are given in Table I in italics. It can be seen that even though the method of cletermination is completely different, the values of the constants for the ML and MHL complexes are in line with those obtained by the method of competition with the proton. For the MH, L and M,L complexes, the values are lower, but it should be noted that both these species correspond to minor species in regard to the previous ones (see Fig. 1). protonation constants, are reported in The values of log K,, stepwise conditional Table 3 and Figure 2. The values determined in the same medium by MLNIQUAD are given in parenthesis. The agreement between both sets of values is good. The comparison between curves (a) and (b), corresponding to the But,NBr and KC1 media shows the strong competition between the potassium and the proton. The effect is especially high for the protonation step 1 and 2, i.e., pH of 6-9, and for supporting-eIectrolyte concentrations ranging from O-O.2 M. Both these conditions are encountered in biological media. It still appears that the influence of alkali-cations must be taken into consideration in both biological and physico-chemical reactions involving inositol-phosphates.
0
FIGURE 2. Curves of the variation of the stepwise protonation constants vs the concentrations of supporting-electrolyte. (a) But,NBr Media; rb) KC1 Media.
-_
l -? . . . .. . supporllnp-tlcctmlyL
(II)
44
Ei. Bieth
et al.
The authors are grateful to PERSTORP PHARMA (Sweden) for providing Ins(l.t,6)P,, and thank Miss Caroline Dietrich for her technical assistance.
the
REFERENCES 1. J. M. Lehn, in Structure and bonding, J. D. Dunitz, Ed., Springer-Verlag. New York, 1973. Anal. Chem. 57, 2956 (1985). 2. P. G. Daniele, C. Rigano, and S. Sammartano, 3. P. G. Daniele, C. Rigano, and S. Sammartano, Talanta 30, 81 (1985). 4. S. Capone, A. De Robertis, C. De Stefano, S. Sammartano, R. Scarcella, and C. Rigano. Thermochim. Acra 86, 273 (1985). 5. A. De Robertis, C. De Stefano, S. Sammartano, R. Cali, R. Purrello, R. Scarcella, C. Rigano, J. Chem. Research (S). 164 (1986); J. Chem. Research (M), 1301 (1986). 6. A. Casale, A. De Robenis, C. De Steiano, Thermochim. Acta 128. 261 (1988). Talanta 34(13), 233 (1987). 7. A. De Robertis, C. De Stefano, S. Sammartano, 8. H. Bieth and B. Spiess, J. Chem. Sot., Faraday Trans. I 82, :935 (1986). 9. H. Bieth, P. Jost, B. Spiess, and C. Wehrer. Anal. Lett. 22(3), 703 (1989). 10. H. Bietb, P. Jost, B. Spiess, J. Inorg. Biochern., in press (1989). 11. M. J. Bet-ridge and R. F. Irvine, Nuture 306, 67 (1984). 12. M. 3. Berridge and R. F. Irvine, Nature 312, 315 (1984). 13. M. J. Bet-ridge, Biochem. J. 220, 345 (1984). 14. Ata A. Abdel-Latif, PhQrmaco~ogica~ Reviews 38(3), 227 (1986). 15. R. Parthasarathy and F. Eisenberg, Biochem. J. 235, 313 (1986). 16. J. W. Putney, D. L. Aub, C. W. Taylor, and J. E. Merritt, Fed. Proc. 45, 2634 (1986). 17. B. W. Agranoff, Fed. Proc. 45, 2629 (1986). 18. D. Lichtstein and D. Rodbard, Ltye Sciences 40, 2041 (1987). 19. M. J. Berridge and R. F. Irvine, Nature 341, 197 (1989). 20. M. J. Vallejo, T. Jackson, S. Lightman, and M. R. Hanleyt, Nature 330, 656 (1987). composition and a method for preparing inositoltrisphos21. Siren Matti, A pharmaceutical phate, U.S. Patent No. 4735936. 22. P. F. Worley, J. M. Baraban. S. Supattapone, V. S. Wilson, and S. H. Snyder, J. Biol. Chem. 262, 12132 (1987). 23. A. Sabatini, A. Vacca, and P. Gans, Tafunta 21, 53 (1974). 24. A. De Robertis, C. De Stefano, S. Sammartano, and C. Rigano. Talanta 34(11). 933 (1987). 25. P. Jost and B. Heulin, L’actualite chimique, Avril, 34 (1984). 26. L. 0. Sillen. A. E. Mat-tell, Stability constants of metual-ion complexes, Special publications No. 17 (1964) and No. 25 (1971), ChemicaI Society, London. 27. S. T. Rao and J. Sundaralingam, J. Am. Chem. Sot. 91, 1210 (1969). 28. C. A. Beevers and G. H. Maconochie, Acta Cryst. 18, 232 (1965). 29. J. Botts, A. Chashin, and L. Young, Biochemistry 4, 1788 (1965). 30. B. A. Klyashchitskii, V. 1. Shvets, and W. A. Preobrazhensky. Chem. Phys. Lipids 3, 393 (1969). 31. V. I. Shvets and R. P. Evstigneeva, Zhur. Org. Khemi 8, 1550 (1972).
Received April 4, 1990; accepted April X9, 1990
