Ternary complex formation between Al(III)-adenosine-5′-phosphates and carboxylic acid derivatives

Ternary Complex Formation between Al(III)-Adenosine-5 ‘-Phosphates and Carboxylic Acid Derivatives

Tam& Kiss, Imre S6v6g6, R. Bruce Martin, and Jouni Pursiainen

TK, IS. Department of Inorganic and Analytical Chemistry, Kossuth University, Debrecen, Hungaly.-RBM. Chemistry Department, University of Virginia, Charlottesville, Vkginia, U.S.-W. Department of Chemistry, University of Oulu, Oulu, Finland

ABSTRACT

The possibility of ternary complex formation has been studied in the Al(III)-adenosine-5’-phosphate (AMP, ADP, and ATP)-ligand B (oxalic acid, lactic acid, and malic acid) systems by pH-potentiometric and 31P NMR methods. Formation of ternary complexes AL4EIH and AlAB is favored in all systems in the acidic pH range. Under physiological conditions at pmolar A13+ concentrations and at high ligand excess, Al(II1) is bound mainly to the nucleotides: almost exclusively in the presence of the relatively weak bidentate Al(II1) binder oxalic acid and lactic acid, and about 30% in the presence of the much stronger tridentate-coordinating molecule, malic acid.

INTRODUCTION

Soluble aluminum species have been implicated in a number of toxic biological processes in aquatic life and plants and humans. Al(II1) is involved in several pathological disorders in humans, including bone diseases and dementias, possi- bly including Alzheimer’s disease. Although the mode of Ah110 action is uncertain, it may be due to the binding of Al(II1) to biophosphates such as nucleic acids, phosphorylated proteins, and nucleoside phosphates [l, 21. Nu- cledtides occur throughout the body and most reactions taking place in living systems involve ATP. Al(II1) binding to DNA is much weaker than that to nucleotides due mainly to the large difference in the basicity of their phosphate binding sites [l, 21. Therefore it is more likely that the toxic effect of A13+ relates to its interference in ATP-associated reactions.

Address reprint requests and correspondence to: Tam& Kiss, Department of Inorganic and Analytical Chemistry, Kossuth University, H-4010 Debrecen, Hungary.

Journal of Inorganic Biochemistry, 55,53-65 (1994) 53 0 1994 Elsevier Science Inc., 655 Avenue of the Americas, NY, NY 10010 0162-0134/94/$7.0-O

54 i? Kiss et al.

Previous spectroscopic studies using 27Al ” P, and ‘H NMR found various Al(III)-ATP (and other nucleotide) complexes of 1:l composition with predomi- nant Al(II1) coordination at the phosphate moiety 13-51. Recently we have studied potentiometrically the AU111 complexation reactions with adenosine- S-phosphates (AMP, ADP, and ATP) and found bis complexes also to be important in Al(II1) binding 161. Biophosphates are not the only potential Al(II1) binders in extracellular or intracellular fluids as other O-donor containing low molecular weight bioligands can also take part in Al(II1) coordination via the formation of either binary or ternary complexes. Furthermore, these latter complexes can also mimic Al(II1) bound to larger biomolecules such as proteins in biological systems. The interaction of Al(II1) with small molecular weight dicarboxylic acids (oxalic and malonic acids) and hydroxycarboxylic acids (glyco- lit, lactic, malic, citric, and tartar%) has been widely studied [7-171, although the results obtained are sometimes contradictory due to the high hydrolytic tendency of Al(III), which results in slow equilibration. It appears well-established, however, that the ionization of the hydroxyl group of coordinated hydroxycarboxylate ligands starts at pH N 3. Besides monomeric species hydroxo- and oxo-bridged polynuclear, in some cases highly polymeric, complexes are also formed readily. The extent of polynuclearity seems to decrease with the increase in the denticity of the ligands.

Little attention has been given to ternary systems including nucleotides and hydroxycarboxylic acids. In a recent study 1181 ternary complexes in the Al(III)- ADP-F system were found with a frequency predicted statistically on the basis of binary complex stabilities. There is the possibility that an appropriate ligand tightly bound to Al(II1) would form a complex that resists hydrolysis and permits studies with second ligands.

The present paper reports the results of a pH-potentiometric and partly 31P NMR spectral study on the speciation of complexes formed in the Al(III)- adenosine-S-phosphate (AMP, ADP, AI?)-carboxylic acid (oxalic, malonic, lactic, and malic acids) ternary systems. Moreover, malonate provides an excellent model for the y-carboqglutarnate side chain that occurs in some proteins.

EXPERIMENTAL

Reagents

Adenosine-S-phosphates were the best quality sodium salts available from Sigma Chemical Co., while the dicarboxylic acids and hydroxycarboxylic acids were Reanal products of puriss quality. Their purities were checked, and when possible the exact concentrations of their solutions were determined by the Gran method [19]. The A13+ stock solution was prepared from recrystallized AlCl, X 6H,O, and its metal ion concentration was determined gravimetrically via its oxinate. The ionic strength of all solutions studied was adjusted to 0.2 mol dmp3 KCL In all cases the temperature was 25.0 + O.l”C.

pH-Metric Measurements

The stability constants of the proton and Al(IH) complexes of di- and hydroxycarboxylic acids were determined by pH-metric titrations of 25 cm3 samples. The concentration of the ligand was 0.008,0.004, or 0.002 mol dmV3 and the metal

TERNARY COMPLEXES OF AL(III)-NUCLEOTIDES 55

ion-to-ligand molar ratios were O:l, 2~1, l:l, 1:2, 1:3, 1:4, 1:6, 18, or 1:lO and l:l:l, 1:1:2, 1:2:1, or 1:2:2 for the binary and ternary systems, respectively. The titrations were performed over the pH range 2.2-8, or until precipitation occurred with KOH solution of known concentrations @a. 0.2 mol drnp3) under purified argon atmosphere. In the absence of ligand excess the samples became opalesque at pH N 5, while in the case of some excess of ligand the solutions remained clear up to pH N 7-8. Above pH N 5 about 5-15 min or more were required to reach pH-equilibrium. The reproducibility of the titration curves was within 0.010 pH unit throughout the whole pH range. Titration points, when equilibration could not be reached in 10 min, were omitted from the calculation. For the hydroxo complexes of Al(III), the stability constants (log p> assumed [20] were -5.52 for AlH_12+, -7.70 for A12H_24+, - 13.57 for Al3H_45+, - 109.1 for A113H_327+, and -23.46 for AlH_,-.

The pH was measured with a Radiometer PHM 84 instrument with a GK2322C combined glass electrode which was calibrated for hydrogen ion concentration according to the method of Irving et al. [21]. The concentration stability constants PPqT = ~~,~,~,l/~~lP~~lq~~lr were calculated with the aid of the computer program PSEQUAD [22].

“P NMR Measurements

NMR spectra were recorded on a Brucker AM-200 spectrometer at 25°C. Chemical shifts are referenced to the signal of 85% orthophosphoric acid used as an external standard. Aqueous solutions of the samples contained 20% of D,O to provide an NMR lock signal. Typically, 500 scans were accumulated per spectrum.

RESULTS AND DISCUSSION

AI0IIl-Oxalic Acid and ARIB)-Malonic Acid

The basic coordination in both systems, as confirmed by NMR measurements 191, is mono, bis, and tris complexes via (COO-, COO-) chelation. The higher stability of the 5-membered chelate rings of oxalic acid complexes compared to the 6-membered rings of malonic acid complexes is clearly reflected in the basicity-adjusted stability constants log K,, - log &r2A, which are 3-4 log units larger for the Al(II1) complexes of oxalic acid. Neither of these two dicarboqlic acids can, however, keep Al(II1) in solution around neutral pH; Al(OH), precipitates at pH * 7 in the Al(III)-oxalic acid and at pH N 6 in the Al(III)- malonic acid systems even at a three-or fourfold excess of ligand. The stronger coordination of the oxalate ion to A13+ allows detection of various mixed hydroxo species before precipitation, while those are rare in the Al(III)-malonic acid system because hydroxyl ion, being a stronger competitor to malonate, can more readily displace coordinated malonate, which leads to a easier precipitation of AI(O The time allowed between pH-meter readings has a bearing on the complexes formed as shown in Table 1.

The differences originate from slow hydrolytic reactions. In our case, when more than 10 min were necessary to reach pH equilibrium ( f 0.002 pH changes in 2 min) the titration points were omitted from the calculation. Under these circumstances titration curves were best fitted with the monomeric species given

56 T. Kiss et al.

TABLE 1. Proton (pK) and Aluminum (III) (log /3) Stability Constants for the Com- plexes of Dicarboxylic Acids at 25.0 f O.l”C

Species Ref. 7a

pK(Hd 3.51 pK(H,A) 1.07

Methyl

Malonic Malonic Oxalic Acid Acid Acid

Ref. 8b Present’ Present’ Ref. 10b

3.57 3.730) 5.150) 5.09

0.97 1.08(9) 2.59(2) 2.77

AL4

‘4lA2

‘4l‘43

m-1

m-2

fi,H-1

M,A,H- 3

&AJ-z

- 5.94 - - -

6.06 5.97 6.2205) 6.23(2) 5.65 11.09 10.93 11.37(4) 10.92(2) 10.00 15.12 14.88 15.40@) 13.39(6) 12.40

- - - 1.04(4) -

- - 4.385) -5.35(9) - - - 5.31(9) - -

- - 9.34 - -

- 4.46 - -

MKAIA/KA,A~) 1.03 1.01 1.07 1.54 ~~~~KMAJKAIAJ 1.0 1.01 1.12 2.22 IogK,A - PBH~A 1.42 1.43 1.41 - 1.51 logK.u, - PPH~A 0.39 0.42 0.34 - 3.05 IogK,AS - P@H*A -0.61 - 0.60 - 0.78 -5.52

’ In 1.0 mol dmm3 NaClO,; b in 0.6 mol dmm3 NaC1, ’ in 0.2 mol dme3 KCl.

1.30 1.95

-2.21 -3.51 -5.46

in Table 1. Oligomeric complexes like L!&A~H_~, Al,A,H_, detected by SjSberg et al. [8] were rejected by the computer program even if their stoichio- metric compositions are (AlAH_, )3 and (AI&H_ 1)2, whole number multiples of our monomeric species. The different speciation models result from different time scales for pH-meter readings. In the solution equilibrium group at Umei University true pH-equilibrium is measured independently of the time necessary to achieve it (sometimes it takes hours), thus on the one hand, a wider pH range is used to describe the system, and on the other hand, the very slow olation/oligomerization reactions not followed by us are also taken into ac- count. When the time scale and ionic strength differences are accounted for, there is excellent agreement between the two studies.

AI(III)-Lactic Acid and ANIID-Malic Acid

These hydroxycarboxylates bind AN110 more weakly than the simple dicarbox- ylates since alcoholic hydroxyl, especially in protonated form, is a weak coordination site as compared to a carboxylate. Hence, besides the (COO;OH) coordinated complexes (ALA, type complexes for lactic acid and AlAH for malic acid), which are formed at pH < 3, various deprotonated species are the dominant complexes. Protonated alcoholic-OH probably participates in the coordination as the stability of these complexes is about 1 log unit larger than that of the corresponding propionate and malonate complexes [23]. The ratio of the step- wise stability constants is lower or only a little higher than 0.6 log unit (see Table 2) expected on the basis of the purely statistical case, which is rather

TERNARY coMpLExHs OF AL(III)-N~CLE~TIDES 57

surprising as no change in the octahedral coordination geometry can be assumed in the AlAH, type complexes. Formation of (COO-,OH) type bis and tris complexes is negligible, indicating that the solid compound Altlactate), with six-coordinated octahedral geometry 115, 161 does not persist in aqueous solutions. In the pH range 3-5 complexes of AlAH_, and Al&H_, are the dominant species in both Al(III)_lactic acid and Al(III)-malic acid systems. In these complexes the likely metal ion induced deprotonation of the alcoholic-OH and alcoholate-O- coordination is suggested by a comparison of the stability constants with those of related compounds [12, 131, and largely confirmed by NMR (‘H, 13C, and 27Al) techniques 116, 171. Spectroscopic results support formation of alcoholate-O- coordinated complexes in this pH range; however, proton dissociation from a coordinated water molecule and parallel formation of mixed hydroxo complexes AlA and AlA, cannot be completely ruled out. These pK values are 4.32 and 3.50 for lactic acid, much lower than the pK = 5.5 of a water molecule in the /L~(H~O&~+ ion, indicating either proton liberation from the ligand molecule or a reduction in coordination number during ionization of a water molecule [24]. In these systems it is likely that formation of similar bonding isomers occurs much more frequently in solution than mentioned in the literature.

TABLE 2. Proton (pK) and Aluminum(II1) (log 8) Stability Constants for the Com- plexes of Hydroxycarboxylic acids at 25.0 f O.l”C

Species

Lactic Acid Malic acid Ref. 13a Present b Present b

pK(H-4 pK(H,A)

AlA

m2

m3

--I

--2

m-3

A&H-l

AzM-2 A,AH_,

G%H-,

43Ad-3,

~Kw./KAIA~)

‘%‘&IA~/&A~)

l0gK.u~~ - P@H~A logK,A - P~HA PKAIA P~IAH_,

PKAIA, AlAl-, +A

3.57 -

- 2.36

4.42 5.79

- -

0.94 -3.29 -

- 9.62 -92.6

0.30 0.41 0.69 0.67

- 3.48

-

3.520) -

- 2.48(2)

4.55(6) 5.95 (15)

- 1.8401) -

- 12.39(g) 1.05(5)

- 3.43(6) -

- 9.60(2) - 96.97(9)

- 1.04 4.32

-

3.50 2.89

4.57(l) 3.16(l)

7.349) 3.84(22)

- -

1.320) - 3.47(3)

-

4.74(2) -

- 3.84(5) -

-

- -

- 0.39

2.52 4.79

-

3.42

a In 0.6 mol dmm3 NaCI; b in 0.2 mol dmm3 KCI.

58 T. Kiss et al.

As the stability constants in Table 2 indicate, deprotonated complexes are much more stable with malic acid, where the presence of another carboxylate group in /3 position allows tridentate coordination of malate via the formation of a 5 + 6-membered joint chelate system. The complex AlAH_, is formed by the liberation of two protons in a highly cooperative way from the P_COOH and the alcoholic-OH groups of the ligand (pK,,, = 3.5 and pK,,, = 2.52). In the species A&H_ 1 only one ligand molecule is coordinated via deprotonated alcoholate: the second molecule is bound in a (COO-,OH) manner. Accord- ingly, the equilibrium constant (1ogK) of its formation process AlAH_ 1 + A = A&H_ 1 is 2.89 for lactic acid and 3.42 for malic acid.

Following the recent observation of Marklund and &man [13], we found that the fit of the experimental titration curves improved by about 30%, when formation of oligomeric hydroxo species (Al,A,H_, and Al,,A,H_,,) were also assumed in the Al(III)-lactic acid system. Such oligomeric species might also be important in high metastability of the aqueous solution of Al(lactate), [16]. In the Al(III)-malic acid system the various oligomeric mixed hydroxo species such as Al,A,H_,, Al,A,H_,, Al,A,H_,, Al,A,H_,, Al,,A,H_,,, etc. reported [12, 13, 25-291 to be likely formed in various Al(III)-hydroxycarboxylic acid systems were always rejected by the computer program during calculation. A monomeric mixed hydroxo species AlAH, (or more precisely Al@I-I_ 1 XOH)) seems to be dominant at pH > 6 in this system.

AI(II&Adenosine-5’-Phosphates

Our recent solution speciation study [6] on Al(III)-adenosineJ’-phosphate (AMP, ADP, ATP) systems revealed that A13+ readily forms equimolar complexes with adenosine-nucleotides, which are stronger than those of most metal ions. In addition to the 1:l complex, a bis complex is formed which dominates in slightly acidic solution. In neutral solution mixed hydroxo complexes also form, then OH- ions displace the coordinated nucleotides, and the very stable Al(OH),- is formed in basic pH range. Previous 31P NMR studies [4] assumed formation of mainly equimolar complexes from the appearance of the NMR resonance of the free noncoordinated nucleotide molecule at any excess ligand.

However, as is shown in Figure 1 at least 30% of the nucleotide is in free form in the metal complex formation pH range between 3 and 7 at 1:2 metal-to-ligand ratio, even if formation of bis complexes is also assumed, which can be easily detected by NMR. In the adenosine5’-phosphate series the log K,, characteristic of binding of Al(II1) to the completely deprotonated phosphate groups shows a sequence of AMP < ADP < ATP, suggesting Al(II1) chelation to the two terminal phosphate groups in ADP and ATP and coordination to the single phosphate in AMP.

AI(II&nucleotide-B Ligand Ternary Systems

Mixed ligand complexes of composition AlABH, AlAB, and AlABH_, were assumed in the computer fitting of the experimental titration curves obtained at different metal ion-to-ligand ratios. Based on the Al(II1) binding ability of the nucleotides in their binary complexes coordination of the terminal phosphate donor(s) can also be assumed in the ternary complexes. Thus, only the bonding mode of ligand B can change in the mixed ligand complexes protonated to different extents. Protonated complexes were formed only with ADP and ATP

TERNARY COMPLEXES OF ALtIII)-NUCLEOTIDES 59

c .103

mol ldn? I

15

10

5

I

A/.

3 4 5 6 7 PH

FIGURE 1. Speciation curves, mol dmP3 concentration versus pH, for Al(III)-ATP system. C, = 0.001 mol dmP3, C, = 0.002 mol dme3.

independently of the capability of the other ligand to form protonated complexes. With oxalic acid and lactic acid, which do not form protonated species, the proton can be bound only at the base-N of the nucleotide. With malic acid, however, an equilibrium of two isomeric species can be assumed: one protonated at the nucleotide base-N and the other protonated at the pcarboxylate of malic acid. Our pH-metric measurements cannot differentiate between the two species. Deprotonation of the complex AlABH can take place either with or without structural rearrangement of the coordinated molecules. In the latter case the proton is liberated from the noncoordinated nucleic base and/or the p-car-boxy1 of malic acid, while in the former case the coordinated alcoholic-OH of lactic acid and malic acid lose a proton, forming a (COO-,O-) bonding mode. A comparison of the pKAIAaH values with the pKbase_n and pK,,, values shows that they are closer to the pK value of the nucleic base-N, suggesting that the proton is liberated from the noncoordinated base donors without any change in the coordination sphere of the Al(II1).

Formation of ternary complexes are little favored between nucleotides and oxalate (A 1ogK values are all rather negative), although binding of a nucleotide molecule to the binary complex of oxalic acid AlB is generally more favored than binding to its own binary complex AlA. This tendency can be explained by the less steric hindrance and electrostatic repulsion between the ligand molecules in the ternary complex compared to the 1:2 binary complex Al-nucleotide.

As can be seen in Tables 3 and 4 this is true for the mixed complexes of the hydroxycarboxylic acids too. Namely, the strength of nucleotide coordination to the (COO-, OH)-coordinated Al(lac)*+ or Al(malH)*+ is about the same and somewhat larger than to Al(ox)+ due probably to the effect of charge neutral- ization. At the same time binding of a nucleotide molecule is less favored to the (COO-, O-)-coordinated uninegative AMacH _ 1 1’ and similar to Al(ox)+. Again electrostatic reasons seem to be primarily responsible for the stability enhance- ment of the ternary complexes. The equilibrium constants of the Al(mal)+ + A reaction are between those characteristic of the reactions between nucleotides and the (COO-, OH)-coordinated or (COO-, O-)-coordinated hydroxycarboxyl-

60 T. Kiss et al.

TABLE 3. Stability Constants of Mixed Ligand Complexes of Adenosine-5’-Phosphates (A) with Carboxylic Acid Derivatives (B) at 25.0 f 0.1 and I = 0.2 mol clrne3 WC0

System

AlABH

log B A log K* A log K*

NH-,

1% P PK,,

Oxalic acid

AMP AJIP ATP

Lactic acid AMP ADP

ATP

Malic acid AMP

ADP ATP

- 10.40(14) -1.99 - 15.48(17) - 1.72 11.88(g) -2.16 - 16.24(16) - 1.28 12.57(3) -1.57 -

- - 8.45(3) - 0.20 2.8(3) 5.6 13.86(4) 0.40 9.99(6) -0.31 4.5(2) 5.7 13.95(9) 0.17 10.46(8) 0.06 5.0(2) 5.5

13.53(5) 0.02 9.48uO) - 0.53 -

15.08(4) - 0.08 11.08(8) -0.58 - - 15.52(g) 0.26 11.29(9) -0.47 - -

*AlogK = log %I,,(,, - ~%KAIA(H, - log K,,; A 1ogK > -0.4 for octahedral geometry, and A log K > - 0.6 for tetrahedral geometry indicates that formation of the ternary complex is favored

[301.

ate complexes of AKIII), confirming the assumption that two binding isomers being in equilibrium can be ascribed to the complex Al(mal)+.

The A 1ogK formulation bases the tendency toward mixed complex formation on 1:l complexes. The A 1ogK represent the equilibrium constant for the reaction MA + MB = MAB + M. On this basis, formation of the mixed complexes for any case in Table 3 is not especially favored beyond a reasonable statistical value. A different picture emerges when mixed complex formation is based on 1:2 complexes according to MA, + MB, = 2 MAB. Regardless of coordination geometries, the statistical value for the equilibrium constant re- mains X = 4 (1ogX = 0.6). From the equilibrium constants in Tables 1, 2, and 3 we find the following 1ogX values: oxalate, 1.30 and 0.23 for ADP and ATP; lactate, 3.90 and 3.27 for ADP and ATP, respectively. Thus, with lactate the mixed complex is favored by a factor of 500 and 2000 beyond the statistical value in comparison with binary 1:2 complexes.

It is surprising that ternary hydroxo complexes AlAB or AlA(BH_ 1 XOH) are not formed in any of the systems studied. In the Al(III)-nucleotide-lactate

TABLE 4. Derived Equilibrium Constants Characteristic of Ternary Complex Formation

Process

A=

AMP2_ ADP3_ AT+

Al+A 6.17 7.82 7.92 AlA-kA 4.18 4.34 4.55 ANox) + + A 4.18 5.66 6.35 Al(lac)*+ + A 5.97 7.51 7.98 Al(malH)*+ + A 6.19 7.74 8.18 Al(lacH_,)++A 4.64 6.24 6.74 Al(mal)+ + A 5.64 7.24 7.45


s % 0.6

E! AIAB

; 0.4 z

0.2

Ii

3 4 5 6 PH FIGURE 2. Speciation curves, mole fraction of A13+ versus pH, acid system at 1:2:2 ratio; C, = 0.001 mol dmm3.

-2

for AI(III)-ATP-oxalic

systems the bonding mode of complex AlABH_, cannot be decided unambigu- ously, as the pK,,, values allow the assumption of deprotonation of either the alcoholic-OH of lactic acid or a water molecule in the coordination sphere of Al(II1). The absence of quatemary hfdroxo complexes might be connected with a reduction in coordination number during hydroxo complex formation. Such a reduction accounts for the cooperativity observed in the Al(III)-OH- binary system [24].

Figures 2-4 show the species distribution for the ATP ternary systems presented as mole fraction of Al(II1) versus pH. As in Figure 2, Al(II1) binds mainly in oxalate binary complexes in acidic solution, where the ternary complexes AlABH and AlAB become the dominant species in the pH range 4-6, and in the physiological pH range the coordinated oxalate is displaced by ATP and/or hydroxyl ion. Against the hydroxycarboxylate lactic acid or malic acid

1.0

t

0.2

t AIAH

3 4 5 6 PH FIGURE 3. Speciation curves, mole fraction of A13+ versus pH, for Al(III)-ATP-lactic acid system at 1:2:2 ratio; C, = 0.001 mol dmp3.

62 T. Kiss et al.

0.8

.E 0.6

e

,” 0.4 “a

0.2

3 4 5 6 PH

FIGURE 4. Speciation curves, mole fraction of A13+ versus pH, for Al(III)-ATP-malic acid system at 1:2:2 ratio; C, = 0.001 mol dme3.

(Figs. 3 and 4), ATP is more efficient in binding Al(II1) at acidic pH. In the physiological pH range, however, malic acid, being capable of tridentate coordination, displaces the nucleotide molecule from the ternary complex. In slightly basic media hydroxyl ion becomes the most efficient Al(II1) binder. It is worthwhile to note that formation of the poorly characterized polynuclear hydroxo complexes is practically negligible in these ternary systems up to pH _ 7. Thus, the obscurity in the speciation of the Al(III)-hydroxycarboxylic acid binary systems fortunately hardly affect the description of the ternary systems.

In order to get more detailed information on the species distribution in the physiological pH range where the information content of the potentiometric data alone are limited because of the poorly controllable olation processes, 31P NMR measurements were also carried out. Due to the relatively slow chemical exchange reactions on the NMR time scale between free and complexed ATP, 31P NMR can be used directly to monitor the state of the nucleotide in its binary and ternary systems [4, 51. Figure 5 shows the 31P NMR spectra of ATP alone, in the presence of equimolar A13+, and equimolar or fivefold excess of ligand B at pH = 7.0. The doublet resonances at - 5 and - 9 and the triplet at - 20 ppm are assigned to the PY, P,, and Pp resonances of ATP, respectively [4, 51. A13+ has a pronounced effect on the spectrum of ATP, including broadening and upfield shifts of all resonances with a close overlap of y- and a-phosphate signals (see Fig. 5B). When oxalic acid is also present in equimolar amount (Fig. 5C) new resonances occur both in the p-phosphate region at -22.5 and in the y, a-phosphate region at - 11 ppm beside the signals of the free and the binary-complexed ATP. These new resonances can be assigned to the ATP bound in the ternary complex AlAB via the terminal p- and yphosphate groups. At higher excess of oxalic acid ATP is completely displaced from the binary Al(III)-ATP complexes (Fig. 5D), and in accordance with the species distribution Al(II1) is primarily bound in the Al(III)-oxalate binary complex AlB, and N 90% of ATP is in uncomplexed form.

Lactic acid seems to be a less effective Al(II1) binder in the presence of nucleotides. Both in equimolar solution of the components and at fivefold lactic


-- I

0 -5 -lb -1; -2b ppm’ FIGURE 5. 31P NMR spectra of binary and ternary Al(III)-ATP complexes at pH = 7.0. (A) ATP; (B) Al-ATP, 1:l; (C) AI-ATP-oxalic acid, 1:l:l; (D) Al-ATP-oxalic acid, 1:1:5; (E) Al-ATP-lactic acid, 1:l:l; (F) Al-ATP-lactic acid, 1:l:S; (G) Al-ATP-malic acid, 1:l:l; (H) Al-ATP-malic acid, 1:1:5.

acid excess, broad, poorly resolved merged resonances of the free and eom- plexed (presumably more than one species) ATP are observed. A well-separated signal, which may be an indication of ternary complex formation, cannot be observed either in the /3- or in the y, a-phosphate regions, although according to the speciation curves 30% of ATP is found in the ternary complex AlABH_, at 1:1:5 metal ion-to-ligand ratio and 10% at 1:l:l ratio at pH = 7.0. When lactic acid concentration is increased from equimolar to a fivefold excess, the ratio of the free and complexed ATP is hardly changed but the relative amount of the species AlA and AlABH_, (or AlAB( increases from 4:l to 23 in favor of the ternary complex. It is reasonable to assume that substitution of water molecules in the coordination sphere of Al(II1) by a weakly coordinating carboxylate and alcoholic-OH groups of lactate will have a minimal effect on the chemical shift of the coordinated nucleotide phosphates. Thus, the upfield shift of the p- and y,a-phosphates is not so sufficient as in the case of the ternary complex with oxalate to result in a well-separated splitting of the resonances of

64 T. Kiw et al.

g 0.6

z

; 0.4 a

3 4 5 6 7 PH

FIGURE 6. Speciation curves, mole fraction of A13+ versus pH, for Al(III)-ATP-malic acid system mimicking physiological conditions; C, = 1 pmol dme3, C, = 1 mm01 dm-3, Cmalic acid = 0.1 mmol dm-3.

complexed ATP, but large enough to produce highly overlapping resonances, which occur as very broad, almost structureless 31P signals (Figs. 5E and 5F).

The potent Al(II1) binder feature of malate is clearly indicated by its effect on the 31P NMR resonances of ATP (see Figs. SG and 5H). Both at 1:l:l and 1:1:5 Al(III)-ATP-malic acid ratios most of ATP is in uncomplexed form, as confirmed by the relatively high intensity of the well-resolved resonances of the (Y-, p-, and y-phosphates at -4.4, -9.4, and - 19.8 ppm. The intensity of the resonances characteristic of the complexed ATP decreases, while that of the free ATP resonances increases as the excess of malic acid is increased, demon- strating that malate coordinated in a tridentate manner displaces the nucleotide from the coordination sphere of AK110 Thus the 31P NMR results strongly support conclusions based on the potentiometric titrations.

When the above discussed speciation models are applied to physiological conditions in order to assess the AK110 binding feature of plasma, the presence of 1 mmol ATP from the 10 mmol total phosphate (inorganic and organic), 0.1 mmol O-donor small molecular weight Al(II1) binder (e.g., lactate, citrate, etc.) and 1 pmolar amount of A13+ was assumed to mimic the plasma concentration of these components. The speciation curves calculated for these conditions reveal that ternary complexes are not prevalent and N 90% of the total Al(II1) is bound to ATP in binary complexes in the presence of oxalic acid or lactic acid, while only _ 30% in the presence of the more potent Al(II1) binder malic acid, which is able to tridentate coordination of Al(II1) (see Fig. 6). The rest of Al(II1) is complexed by ligand B, again mostly in binary complexes. The concentration of the hydroxo complex Al(OH),- is less than 5% at pH = 7.4. Regarding the A13+ ion binding ability of malic acid and citric acid they are presumably fairly similar, as both ligands can coordinate to A13+ in a tridentate manner forming joint chelate systems. Thus, although our results on Al(III)-adenosine-5-phosphate-malic acid systems can presumably be applied for characterizing the speciation of Al(II1) in blood plasma, a detailed potentiometric and NMR study on the Al(III)-adenosine nucleotide-citric acid ternary systems, both ligands being important small molecular weight AI(II1) binders in biological fluids, is in progress.

TERNARY COMPLEXES OF ALtIII)-NUCLEOTIDES 65

lkanhs are due to Mrs. A’. Giincty for her invaluable heZp in the experimental work This publication is sponsored by the U.S.-Hungarian Science and Technology Joint Fund in cooperation with Department of Health and Human Services, U.S. and Ministry of Social Welfare, Hungary under Project No. 182/92b and by National Science Research Fund, Hungary under Project No. OTKA / T7458.

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Received August 2, 1993; accepted August 6, 1993

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Ternary complex formation between Al(III)-adenosine-5′-phosphates and carboxylic acid derivatives