Complexation studies on inositol-phosphates III. Cd(II), Pb(II), and Hg(II) complexes of D-myo-inositol 1,2,6 trisphosphate

Complexation Studies on InositolPhosphates III. Cd [II I, Pb III I, and Hg [II) Complexes of D-Myo-Inositol 1,2,6 Trisphosphate C. Lapp and B. Spiess Laboratoire de Chimie Analytique Illkirch, France

(IUT-ULP),

Fact&! de Pharmacie de Strasbourg,

ABSTRACT The complexation properties of the D-ntyo-inositol 1,2,6 trisphosphate (Ins( 1,2,6)P,) towards cadmium, lead, and mercury were studied in a 0.1 M tetra-n-butylammonium bromide solution at 25°C and in a 0.2 M KC1 medium at 37°C. Mononuclear, polynuclear, and protonated species were found which display higher stabilities than those formed with alkali-earth cations. For the Cd-Ins( 1,2,6)P, system the nature and stability of the complexes were compared with the nucleoside triphosphate-Cd complexes.

INTRODUCTION For several years, numerous works have been devoted to elucidating the biological role of the inositol-phosphates (InsP). Among the various compounds belonging to the class of inositol-phosphates, those acting in the intracellular medium have been especially investigated [l- 111. Thus, it has been shown that the Ins(1,4,5)P, is a second messenger allowing signal transduction by mobilization of intracellular calcium. In addition to this compound, other more or less phosphorylated derivatives [9-111 participate in the complex regulation of the cellular calcium content. In comparison with the very large number of biological and pharmacological works, only a few physico-chemical studies have been published on the inositol-phosphates, mainly concerning phytic acid, the hexaphosphorylated derivative [12-191. In our laboratory we recently studied the protonation constants and interactions with alkali and alkali-earth metal cations of the Ins( 1,2,6)P, [20, 211. Comparison of the

Address reprint requests to: Professor Bernard Spiess, Laboratoire de Chimie Analytique Faculte de Pharmacie de Strasbourg, 74 Route du Rhin, Illkirch, France. Journal of Inorganic Biochemistry, 42,251-266 (1991) 0 1991 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas,

(IUT-ULP), 251

NY, NY 10010

0162-0134/91/$3.50

258

C. Lapp and B. Spiess

protonation constants determined in the presence and in the absence of K+ showed that the potassium cation strongly competes with the proton of the acidic phosphates, indicating the formation of Ins(1 ,2,6)4,-K complexes. The latter complexes and those formed with Li+, Na+, Rb’, and Cs+ were also studied and showed high stabilities for such kind of cations 12 I J. In addition to the previous cations. which are normal constituents of biological media, there are other cations which may also he involved in complexation reactions in the organism. The attention of dietitians and toxicologists has been drawn, for several years, to the slow but regular increase of the concentration of’ heavy metals such as lead, cadmium. ancl mercury in the organism of vertebrates [22]. It is well-known that these metals are eliminated ‘very slowly and thus tend II? accumulate in target tissues. leading to various disorders and diseast_. Their :fTects IW the functioning of the liver, kidney, cardiovascular system, nervous system. spermatogenesis, and even bone tissues are well described 133-301. Moreover, the role 01’ cadmium in cancer induction has been demonstrated f~oranimal5 ~111Icd to the study of the interactions of cadmium with ligands present in genetic material [3 11. ‘I‘hcsc nucleosides, and phosphorylated sugars such as ligands include the nucleotldes, D-ribose 5’..monophosphatc. The nature of the species and their stabrlity conttants a competition between were determined [32--.A5 /. In the case of the thionucleotidcs. cadmium and magnesium cations was reported 1161. Due to the high toxicity of these metals, the search for antidotrh and a more detailed knowledge of their mechanism of action !S of prime importance. The ligands used for this purpose n~ust mobilize the heav\- cations In order to fucilitatz their elimination from the organism. The most effective liganda known are thiocarbnnatr derivatives. N-substituted glucosamines. 2 ..3-climcrcaptosuccinlc acid. and ~1~1s complexes of the diethylenetriaminepentaacetic acid 137-S]. As part of the investigation of the properties ol‘ inositol-phosphates on rho treatment of disorders which may partly be due to the action of heavy metala. the complexation proper-tic!, of the D-rnyo-inositol t .2,6 trisphosphatc with Cd’ ‘. Ph2 _ I and Hgzi were studied, Indications about the interaction< vlhich may cxisf berwecn these cations and other 1nsP3 are also expected. Because alkali cations compctc with the heavy metals mentioned above, the choice of the medium of study governs both the nature of the species and the stability of the complexes. .2s in the previous work3 [ZO, 211, different media and temperature conditions were ubed; ;I[ 57°C 11’1the presence of 0.2 M potassium, the medium is close to that encountered in the cei!: and at 25°C in the absence of potassium. the results better describe the “intrinsic” complexing properties of the ligand.

EXPERIMENTAL Materials Hydrated Na,HIns(l,2,6)P,, provided by Perstorp Pharma (Sweden) was converted into its acidic form as previously described [ZO]. Reagent grade cadmium (II). lead (II), and mercury (II) perchlorates were used. The base used as the titration reactant was either a solution of tetramethylammonium hydroxide or a solution of sodium hydroxide prepared in carbonate-free water. The concentration of the ligand was determined from the potentiometric titration curve

COMPLEXATION STUDIES ON INOSITOL-PHOSPHATES

259

and the concentrations of the metal salts were obtained by complexometric titration with EDTA. The ionic strength was kept constant by adjustment with potassium chloride, tetra n-butylammonium bromide (But,NBr) or tetraethylammonium perchlorate (Et,NClO,), depending on the medium. All the precautions taken in the experiments were the same as those indicated for the previous studies [20, 211.

Potentiometric

Measurements

The method of competition

with the proton was carried out in the investigation involving Cd’+ and Pb’+. In the case of Hg2+ an auxillary ligand has, in addition, been used. Mixtures of metal and ligand in millimolar concentration ranges and in ratios ranging from 0.3 to 1.2 were titrated with a base. For Hg2+, the auxillary ligand, Cl-, was brought by addition of tetra n-butylammonium chloride at a concentration of 5. 10m4 mol. dmm3. The titrations were performed in 10 cm3 solutions at either 25 or 37°C f 0.1 “C under a nitrogen atmosphere. The pH measurements (pH means the cologarithm of the H+ species concentration) were made with an Ingold combined glass electrode (HA 265) connected to a. Tacussel Isis 20 000 pH meter. This pH meter is a part of an automatic titration device monitored by an APPLE II micro-computer [40]. The standard liquid in the reference compartment of the electrode was replaced in order to minimize the junction potential by the following solutions. Solution 1: 0.01 mol. dmm3 Et,NCl, 0.09 mol. dmp3 But,NBr for the study in the 0.1 M But,NBr medium at 25°C (medium 1). Solution 2: 0.2 mol. dm- 3 KC1 for the study in a 0.2 M KC1 medium at 37 “C (medium 2). Solution 3: 0.1 mol. dmp3 Et,ClO, and 0.005 mol. dme3 Et,NCl for the study in a 0.1 M Et,NClO, medium at 25°C (medium 3). The junction potentials were determined as previously indicated [41] with solutions of p H 2 and 3. The values of the constants a and b obtained in the different media and used for the correction of the pH measurements are noted as follows: medium 1, a = 0.125, b = - 13.10; medium 2, a = 0.027, b = -2.71; medium 3, a = 0.231, b = -23.12.

Constants Refinement The overall protonation constants of the ligand which refer to the general equilibria:

must be taken constant in the refinement of the complexation constants. The values previously obtained [20] for media 1 and 2 are given in Table 1. Since medium 3 differs only slightly from medium 2, only small variations of the protonation constants would be expected; thus, the same constants were used. The complexation constants ox,_ reported here correspond to the equilibria:

xM2++

yH++

zL6-GMXHyL~Z-2x-y)-.

The extent of the complex formation and the hydrolysis of the cations and indications about the nature of the species were deduced from the ii curves as a

25

31

25

But, NBr 0.1 M

KCI 0.2 M

But,NBr

Hgz+

--_

3

2

2 3 1

1 1

2 3

1 1

2

1 1 1

2 3

1

0

0

0 0

1 2 3 1 2 3 4 0 1 0 0 1 2 0 0 1 0 0 0 1 0 0 1

0 0 0 0 0

y -l___l-

x

1 1 1

1

1

I 1

1 1 1

I 1

1 1 1 1 1 1 1

Z

20.92 k 0.37 25.54 * 0.05

0.37 0.04 0.21 0.03

f f f f

16.92 18.48 21.34 18.75

2.6-8.

3.1-7.1

2.9-8.4

f 0.06 *0.13 f 0.04 f 0.06

0.02 0.03 0.11 0.03

13.31 11.85 17.67 10.57

rt f f i

3.6-8.3

3.1-8.2

13.61 18.73 9.53 6.46

0.01 0.01 0.01 0.02

14.67 f 0.04 11.20 f 0.07 6.12 f 0.02

f f f f

2.2-9.9

16.70 22.40 24.80 4.39

4.4-8.8

8.22 f 0.04

i

_- pH Range

14.60 f 0.01 19.81 f 0.01 9.48 f 0.01

log p,,, f u

The stoichiometry of the complexes is grven according to the general formula rndicated in the text. S and K correspond, residuals and the R factor given by MINIQUAD.n Is the total number of observations used for the calculations.

Et, NClO, 0.1 M

25

Pb2+

25

But,NBr

0.1 M

Pb2f

37

Cd2 +

Cd2 +

H+

H+

I Cation

KC1 0.2 M

0.1 M

37

--

t(T)

KC1 0.2 M

Medium

respectively,

266

118

202

127

215

369

113

n

0.0036

0.0077

0.0047

0.0059

0.0065

0.0016

0.0051

R

to the standard deviation of the

6.50 IO-”

1.29 lO-5

6.90 1O-6

8.07 l0-h

8.65 10-6

6.70 10-h

6.55 1O-6

S

TABLE 1. Logarithm of the Overall Stability Constants of the Complexes Formed by Cd*+, Pb’+, and Hg2* with Ins(l,2,@P,

s 8

COMPLEXATION

function

of the logarithm

STUDIES

ON INOSITOL-PHOSPHATES

261

of the free ligand concentration:

C, - [L6-]

- $

[HyL++]

jj= C&z The ii curves corresponding to the Cd-Ins(l,2,6)P, and Pb-Ins(l,2,6)P, systems in medium 1 are represented in Figure 1. It can be seen that these curves are not superimposable for the different metal-to-ligand ratios, indicating the possible presence of polynuclear complexes. For lead, the values of ii lie between 0.5 and 0.6 over a large - log L range. The mean number of ligands bound per metal is about 0.5, thus, a stable homobinuclear complex should be the main species over a large pH range. Taking into account the indications given by the ii curves, the data were then subsequently refined by the program MINIQUAD [42]. In the computational approach the best model was first worked out for each metal-to-ligand ratio, and after obtaining satisfactory results, a final refinement was made. During the data processing, the protonation constants were held constant and the pK, values were taken equal to 13.77 (media 1 and 3) 1431 and 13.20 (medium 2) [20]. The determinations concerning Hg 2+ were carried out differently from those of Cd’+ and Pb’+. The former cation forms sparingly soluble complexes at low pH which are replaced by more soluble species when pH is increased. In order to avoid precipitation and to work only in a homogeneous medium, chloride ions were added at a concentration of 5. 10e4 mol. dm- 3 as an auxilliary ligand. This anion forms stable mercury complexes whose stability constants (log @,a, = 6.7; log P,, = 13.2; log fl,e3 = 14.1) [44] were included during the refinement of the Hg*+-Ins(l,2,6)P, system. As the bromide ions are also expected to strongly interact with Hg*+, But,NBr was replaced by Et,NCIO, at the same concentration.

RESULTS

AND DISCUSSION

The nature of the complexes formed between the Ins(1,2,6)P, and Cd*+, Pb*+, and Hg2+ as well as their stability constants are reported in Table 1. Although for cadmium and lead the determination in the absence of potassium was carried out in a medium containing bromides which weakly interact with these cations, the constants should account for the real complexation properties of the ligand such that they can be compared to those of related compounds. In the 0.2 M KC1 medium, only the cadmium and lead complexes were investigated. In such a medium, mercury forms only mercurichlorides and therefore cannot be studied. It can be seen in Table I that the complexes with the studied heavy metals are quite stable. Considering the previous studies concerning the alkali-earth cations [20], it appears that the cadmium complexes are slightly more stable than those of calcium and magnesium, whereas the constants for lead and mercury complexes are higher by several orders of magnitude. Mononuclear, homodinuclear, and trinuclear species, as well as protonated species were found for most of the systems. The main differences between alkali-earth complexes and heavy metal complexes are two-fold. First, heavy metals form M,L species more systematically than alkali-earth cations and second, show a lesser trend for formation of protonated species (especially lead and mercury). Such

262

C. Lapp and B. Spies

Cd-lns(l,2,6)P,

4

5

6

7

8

g

10

11

12

13

-log L

Pb-lns( 1 ,2,6)P3

4

6

8

10

12

14

16

18

-log L

FIGURE

1.

ii Curves

vs

-logL for the Cd-H-Ins~l,2.6~P,

arid the Ph-H-Ins(l.Z.Ci)P,

systems.

results are likely due to the higher affinity for the ligand of these cations which more easily displace the protons of the phosphate groups. In the case of mercury, the model which gave the best convergence criteria does not include an ML complex. It must be kept in mind that the determination was made in the presence of chlorides and that ternary complexes which are likely to form were not considered irr the calculations. The comparison of the results obtained in the presence and the absence of

COMPLEXATION

STUDIES

ON INOSITOL-PHOSPHATES

263

potassium are in line with those found in the alkali-earth cations studies [20], .i.e., potassium at a 0.2 molar concentration greatly influences the stability of the complexes. The differences of the constants increases when stability of the complex increases. For Pb*+ and the ML complex, the difference reaches 4.11. logarithm units. Nevertheless, for this latter cation, potassium does not appear to change the nature of the species. In order to illustrate the relative importance of the complexes and their pH dependence, the distribution curves of the Cd-H-Ins(l,2,6)P, and the Pb-HIns( 1,2,6)P, systems are given in Figure 2. These curves were calculated for metal-to-ligand concentrations in a ratio of one-to-one. It can be seen that complexation of lead starts at a lower pH than that of cadmium. The main species for the former cation is the Pb,L complex whose percentage is around 40% from p H 4 to 7. Only when p H 7.2 is reached does PbL become predominant. The trinuclear and mononuclear protonated species never exceed 10% and can be considered as minor complexes. In the case of cadmium, the binuclear complex is of less importance in regard to the Cd-Ins(l,2,6)P, and CdH-Ins(l,2,6)P, complexes. The CdH,Ins( 1,2,6)P, species was kept on the basis of the calculations but is uncertain due to its low concentration. In order to evaluate the competition towards cadmium between the inositol-phosphates and other biological active phosphate-containing compounds, it would be interesting to consider the metal ion coordinating properties of some nucleosides triphosphates. As most of these compounds are coenzymes in metal-ion dependent enzyme systems, the stability of the complexes of alkali-earth and transition metals were determined. Since cadmium is able to replace zinc in such systems, it has also been often studied. Thus, the comparison will bear only on the cadmium complexes, no constants having been found for lead and mercury. The studies evoked here were done by Siegel et al. [32-351 who recently paid much attention on the stability and structure of metal ion complexes formed with nucleoside phosphates and related compounds. Some of their results are reported in Table 2. It can be seen that the difference in regard to the Ins(l,2,6)P, system is both in the nature of the species and in their stability constants. For the nucleosides only CdL and CdHL species were reported, whereas for the Ins(l,2,6)P,, polynuclear species do exist. The values of the constants cannot be strictly compared since they were determined in different background media. However, for the nucleoside triphosphates, an addition of 0.4 log units to the conditional constants for a 0.1 M sodium-containing medium gives the stability constants free of Na+ competition [34]. By taking this correction factor into account, it is possible to compare the constants of Table 3 to those determined in a 0.1 M But,NBr medium. The comparison then shows that the ML complex of the Ins(1,2,6)P, is about 0.6 log units higher than that of UTP, TTP, and CTP. For the monoprotonated species, the value of log Kfl,referring to the equilibria Cd*++ HL * CdHL better enables the stability of the complexes to be compared. This constant, calculated by the relation system and logKK, = log Pi,, - log PO,,, is equal to 4.13 for the Ins(l,2,6)P,-Ccl 3.02 + 0.15 for the nucleoside triphosphates which were considered (see Table 2) [34]. Taking into account the correction factor due to the presence of the sodium cations, the difference is also about 0.6 log units in favor of the ligand under study. A careful investigation of the stability constants brought Siegel et al. to the conclusion that, excepting the Cd(ATP)2- complex, all the nucleosides are involved in a pure phosphate coordination. A 0.6 log unit increase in the stability constant

264

C. Lapp and B. Spies

2.5

3.5

4.5

5.5

6.5

90

MxHHyL /C

7.5

8.5

PH

Pbz+

80 Ml.

FIGURE 2. Distribution curves of the Cd-H-Ins(l,2.6)P3 plotted against pN. Cy = C& .=-0.001 mol. dm- ‘,

and

Pb-H-Insi

1 ,2.6)P1

species

then holds for the gain of stability arising when a linear triphosphate is replaced by three vicinal phosphates on an inositol ring. These authors pointed out that in a series which included phosphate monoesters and nucleoside monophosphates, the stability of the complexes is governed by the basicity of the phosphates (341. That is, the complexes increase in stability as the basicity increases. Because two phosphate groups of the InsCl .2.6)P, arc more basic

COMPLEXATION

STUDIES

ON INOSITOL-PHOSPHATES

265

TABLE 2. Logarithm of the Overall and Stepwise Stability Constants of the Cadmium Complexes with Various Nucleoside Triphosphates (Ref. 34)

Adenosine triphosphate (ATP) Cytidine triphosphate (CTP) Uridine triphosphate (UTP) Thymidine triphosphate (TTP)

5.34 5.05 5.10 5.09

3.04 3.15 2.89 -

than for the nucleoside triphosphates, a higher stability of the inositol complex is expected and observed. The formation of polynuclear complexes may be due to the fact that the phosphates on the inositol moiety are able to move about freely both above and below the plane of the ring, allowing optimal interactions with one or two more cadmium cations to be attained. In the case of CTP, UTP, and TTP, the coordination requires a partial folding of the phosphate chain that then leads only to mononuclear species. From the above considerations, it can be seen that if both the Ins(1,2,6)P, and nucleoside triphosphates are present at about the same concentrations, the cadmium would be preferentially complexed by the Ins( 1,2,6)P,. This supports the hypothesis that at least some of the biological effects of this ligand [45] can be related to its cadmium binding properties. The authors Ins(1,2,6)P,.

are grateful

to Perstorp

Pharma

(Sweden) for providing

the ligand

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18.

M. J. Berridge and R. F. Irvine, Nature 306, 67-69 (1984). M. J. Berridge and R. F. Irvine, Nature 312, 315-321 (1984). M. J. Berridge, Biochem. J. 220, 345-360 (1984). Ata. A. Abdellatif, Pharmacological Reviews 38(3), 227-272 (1986). R. Parthasarathy and F. Eisenberg, Biochem. J. 235, 313-322 (1986). M. J. Berridge and R. F. Irvine, Nature 341, 197-205 (1989). M. J. Berridge, JAMA 262(13), 1834-1841 (1989). J. P. Heslop, R. F. Irvine, A. H. Tashjian and M. J. Berridge, J. Exp. Biol. 119, 395-401 (1985). S. R. Nakorski and I. Batty, Trends Pharmacol. Sci. 7, 83-85 (1986). R. F. Irvine, A. J. Lotcher, J. P. Heslop, and M. J. Berridge, Nature 320, 631-634 (1986). M. J. Vallejo, T. Jackson, S. Lightman, and M. P. Hanleyt, Nature 330, 656-658 (1987). N. J. Evans, T. J. Jacks, and F. J. McCourtney, J. Food Sri. 48, 1208-1210 (1983). E. Graf, J. Agric. Food Chem. 31, 851-855 (1983). C. J. Martin and W. J. Evans, J. Inorg. Biochem. 28, 39-55 (1986). C. J. Martin and W. J. Evans, J. Znorg. Biochem. 26, 169-183 (1986). C. J. Martin and W. J. Evans, J. Znorg. Biochem. 27, 17-30 (1987). E. T. Champagne, J. W. Robinson, R. J. Gale, M. A. Nauman, R. M. Rao, and J. A. Liuzzo, Anal. Lett. 18(19), 2421-2443 (1985). E. T. Champagne and 0. Hindosa, J. Znorg. Biochem. 30, 15-33 (1987).

266

C. Lapp and B. Spiess

19. H. Bieth, P. Jost, B. Spiess, and C. Wehrer, Analyt. Lett. 22, 703-717 (1989). H. Bieth, P. Jost, and B. Spiess, J. Znorg. Biochem. 39, 59-74 (1990). H. Bieth, G. Schlewer, and B. Spiess, .I. Znorg. Biochem.. 41, 37-44 (1991). J. 0. Nriagu and J. M. Pacyna, Nature 333, 134-139 (1988). P. M. Sivalingham, Toxicol. Environ. Chem. 19, 125 137 (1989). J. Abel, D. Hohr, and H. J. Schurek, Arch. Toxicol. 60. 370-375 (1987). 25. A. Yasutaka, K. Hirayama, and M. Inone, Arch. Toxicoi. 63, 479-483 (1989). 26. G. Togna, N. Dolci, and L. Caprino, Arch. Toxicol. Suppl. 7, 378-381 (1984). 27. M. Carmignani and I? Boscolo, Arch. Toxicol. Suppl. 7. 383-384 (1984). 28. M. Carmignani. P. Boscolo, and P. Preziosi, Arch. Toxrcoi. Suppl. 12, 326-329

20. 21. 22. 23. 24.

(1988). 29. B. R. G. Danielsson,

L. Dencker,

A. Lindgren,

and H. Tjalve,

Arch.

Toxicol. Suppl.

7, 177- 180 (1984). 30. K. Ogoshi, T. Moriyama, and Y. Nanzai, Arch. Toxicol. 63. 320-324 (1989). 31. R. Maximilien and B. Dero, Report CEA R., 5516 E (1990). 32. H. Sigel, K. Scheller, and R. Milbum, Znorg. Chem. 23. 1933-1938 (1984). 33. H. Sigel, Eur. .Z. Biochem. 165, 65-72 (1987). 34. H. Sigel, R. Tribolet, R. Malini-Balakrishnan. and R. Bruce Martin. Znorg. Chem. 26. 2149-2157 (1987). 35. S. Massoud and H. Sigel, Znorg. Chem. 27. 14471453 36. V, L. Pecoraro. J. D Hermes. and W. W. Cleland,

(1988).

Biochemistry

23, 5262-5271

(1984). 37. M. M. Jones, M. A. Basinger, R. J. Topping, G. R. Gale, S. G. Jones, and M. A. Holscher, Arch. Toxicol. 62, 29-36 (1988). 38. G. R. Gale, L. M. Atkins, A. B. Smith, S. G. Jones, M. A. Basinger, and M. M. Jones. Arch. Toxicol. 62, 428-437 (1988). 39. S. G. Jones and M. M. Jones, Environ. Health Perspect. 54, 285.--290 (1984). 40. P. Jost and B. Heulin. L’ActualitP Chimique Avril 34-37 (1984). 41. H. Bieth and B. Spiess, J. Chem. Sot. Faraday Trans. I 82. 1935 ‘-1943 (1986). 42. A. Sabatini, A. Vacca, and P. Gans, Talanta 21. 53-77 (1974). 43. R. Fischer. Thesis Strasbourg (1967). 44. L. G. Sillen and A. E. Martell, Stability constants of metal-ton complexes. The chemical Society. London 17, (1964). 45. M. Siren, Pharmaceutical Composition containing Znositol triphosphate. U.S.Patent No. 4,735.936.

Received September 9, 1990; accepted November 20, 1990