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Ketoglutaric Acid in Acidic Aqueous Solutions

Ketoglutaric Acid in Acidic Aqueous Solutions

ANALYTICAL SCIENCES FEBRUARY 2003, VOL. 19 273 2003 © The Japan Society for Analytical Chemistry

Multimethod Characterization of the Interaction of Aluminum Ion with -Ketoglutaric Acid in Acidic Aqueous Solutions

Xiaodi YANG, Shuping BI,† Xianlong WANG, Jian LIU, and Zhiping BAI

State Key Laboratory of Coordination Chemistry, Department of Chemistry, Nanjing University, Nanjing 210093, P. R. China

It has recently been reported that aluminum plays a very important role in reducing the activity of Krebs-cycle and in rat brain homogenate. Therefore, it is necessary to identify the aluminum binding ability with the pivotal substrate α-ketoglutarate in biological systems. The interactions of aluminum with α-ketoglutarate were studied with pH-potentiometry, cyclic voltammetry, UV-vis, 1H, 27Al-NMR and Raman spectra multi-analytical techniques in acidic aqueous solution to measure the stoichiometries and stability constants of the complexes and its keto- tautomerism. The α-ketoglutarate was found to bind Al in a bidentate manner at the carboxylate and carbonyl + 2+ Ð 3Ð 4+ moieties. The mononuclear 1:1 (AlLHÐ1, AlL , AlHL ) and 2:1 (AlL2 , AlL2HÐ2 ) species, and dinuclear 2:1 (Al2L ) species were found in acidic aqueous solution. Meanwhile, Al can promote α-KG tautomerize to its enolic-structure compounds in solutions. These findings may help to further understand the influence of Al on GDH reactions in biological systems.

(Received May 2, 2002; Accepted September 6, 2002)

and phosphofructokinase.13,15,16 All of these Introduction findings reinforce the possibility that Al could interfere with the pathways in mitochondria. In particular, Al There has been a significant focus on aluminum (Al) chemistry inhibits the reversible reaction of GDH catalyzed converting of to study its role as a toxic agent to organisms.1Ð6 It has been glutamate to α-ketoglutarate. This inhibitory effect could documented that Al causes an inhibition of root formation in decrease the formation of α-ketoglutarate. The concomitant ,1,2,8 abnormal development in fish, neurological and bone inhibition of by Al also further contributes to the tissue and hematological disorders in human.9,11 decreased production of α-ketoglutarate. It could influence on Due to the increased bioavailability of Al and its effects on “Krebs cycle” in brain cells. Moreover, defects in the synthesis life systems, research on determining how Al enters different of bioenergy may play a role in the pathogenesis of cells and affects cell chemistry and morphology, as well as the neurodegenerative diseases, such as AD (Alzheimer’s disease) mechanism of its action and the fate of the Al species are and PD (Parkinson disease).11,14 In addition, the inhibitory essential areas of Al chemistry for the future.7,10 Alpha- effect also affects the formation of glutamate, which may ketoglutaric acid (α-KG, in Fig. 1) is widespread in many influence the synthesis and contribute to the Al microorganisms, and is isolated from higher plants. As an toxicity mechanism of higher plants. intermediate in , it regulates some Since α-ketoglutaric acid plays such a very important role in fundamental physiological processes, e.g. “the Krebs cycle”. enzyme systems, especially in the Krebs cycle and Furthermore, as an intermediate in process, its interaction with Al may influence converted to L- by glutamate dehydrogenase the fundamental process. It will be very interesting to (GDH), it therefore occupies a pivotal position in the investigate the effects of the Al complex of α-KG in order to metabolism of most life forms, and provides a link between further understand Al’s role and mechanism in these enzyme carbohydrate and metabolism.12 reactions. It requires knowledge of Al-α-KG interactions at the More recently, Zatta and his co-workers14 reported that Al has been shown to affect the enzyme activity of the Krebs cycle: Al activates α-ketoglutarate dehydrogenase and . In contrast, aconitase and glutamate dehydrogenase (GDH) showed a decreased activity in the presence of the metal ion of Al in rat brain homogenate. The activity of , a competitor for the substrate isocitrate, appeared to be markedly diminished in cells exposed to Al.21 Furthermore, Al has been demonstrated to be a strong inhibitor to some enzymes of as well as

† To whom correspondence should be addressed. E-mail: [email protected] Fig. 1 Structures of α-KG and its Al(III) complexes. 274 ANALYTICAL SCIENCES FEBRUARY 2003, VOL. 19

Table 1 Proton (log K) and aluminum(III) complex-formation constants (log β) of the hydroxo and α -ketoglutaric acid at 25±0.1ûC and I = 0.10 M (KCl) Ligand Hydroxo Complex log Ka log β24 log βa species species species

2+ 2+ This work AlHÐ1 Ð5.33 AlHL 6.55±0.02 Ð + + HL 4.63±0.02 AlHÐ2 Ð10.91 AlL 3.83±0.03 H2L 2.41±0.01 AlHÐ3 Ð16.64 AlLHÐ1 Ð0.87±0.03 Ð Ð AlHÐ4 Ð23.46 AlL2 5.75±0.05 4+ 3Ð Ref. 26 Al2HÐ2 Ð7.15 AlL2HÐ2 Ð4.58±0.06 Ð 5+ 4+ HL 4.68 Al3HÐ4 Ð13.13 Al2L 6.32±0.03 7+ H2L 2.47 Al13HÐ32 Ð107.41 a. Averages (± standard deviations) for three or four titrations. Fig. 2 Part of the titration data plotted as curves of the pH versus the volume of the titrant for the ligand-to-metal (L/Al) ratios of 1:1, 2:1 and 5:1. CAl = 0.001 M, CKOH = 0.1055 M. molecular level. Such bioinorganic studies were performed in order to clarify various aspects of structures of Al-α-KG complexes, and to fill the gap between knowledge concerning biochemical and biological phenomena. In the past, there were solution to provide an inert atmosphere with stirring. many studies on the interaction of transition metal ions with Additional stirring of the solution was achieved with a magnetic , as well as Cu2+, Co2+, Ni2+, Zn2+, Fe3+, Ca2+, stirrer. The concentration of Al3+ was 1 mM, and the ligand-to- Sr2+ metal-ion interaction with α-KG.17Ð20,25 However, the study metal ion ratios were 1:0, 1:1, 2:1, 5:1 for the binary system, of complexation between Al and α-KG has not been found in respectively. Titration was performed over the pH rang 2.00 Ð the available literature. In this paper, we report on the 10.61, or until precipitation occurred, with a KOH solution of interaction of Al with α-ketoglutarate in acidic aqueous solution known concentration (ca. 0.1 M) and a constant ionic strength (I in order to further understand Al’s potential toxicity in = 0.1 M KCl). Duplicate titrations were carried out and the biological systems. reproducibility of the titration curves was within 0.01-pH unit over the whole pH range. The stability constants of the main metal species were calculated with the aid of the computer Experimental program BEST.23 Complexes were added to the model one at a time until the lowest value of σfit was achieved (usually less Chemical and solutions than 0.02). All chemicals were obtained from Shanghai Chemical Reagent Factory or Beijing Chemical Factory and used as NMR experiments received unless otherwise noted. Alpha-ketoglutaric acid (α- 1H-NMR experiments were operated at 500 MHz on a Bruker KG, 99.50%) was of biological-reagent grade. Its stock solution AM500 spectrometer (Swiss). Al-α-ketoglutarate was prepared

(0.01 M) was prepared daily before experiments by dissolving in D2O to provide all NMR lock signals. The pD values were an appropriate amount of α-KG solid in double-distilled water. calculated via the equation27 pD = pH-meter reading + 0.45 to A stock solution of Al3+ (0.01 M) was prepared by dissolving allow for isotopic and solvent effects caused by the substitution high-purity metallic Al powder (99.99%) in hydrochloric acid of normal water (calibration environment) with heavy water and diluting it to 250 ml. More dilute solutions were prepared (measurement environment). Chemical shifts were referenced by diluting this solution with double-distilled water. To prevent to HDO for 1H-NMR spectra. Typical NMR parameters had a hydrolysis of the metal ion, the Al stock solutions were pulse width of 50 ms (30û) and, a pulse repetition time of 1.29 s; prepared at pH < 2. The ionic strength of a potentiometric- 128 scans were accumulated for the 1H-NMR spectrum. The studied solution was adjusted to 0.1 M with KCl. Potassium 27Al NMR spectra were recorded on a JEOL 90Q FT-NMR hydroxide solutions (ca. 0.1 M) were standardized against spectrometer (Japan) at 23.29 MHz, with 8 K data points and a potassium hydrogenphthalate. Most chemicals were of 2500 Hz spectral width. Chemical shifts were referenced to an 1 27 3+ analytical reagent grade. H, Al-NMR experiments were external coaxial insert containing 0.1 M [Al(H2O)6] included prepared by dissolving appropriate amounts of α-KG and with every sample for the 27Al NMR spectra. The NMR

AlCl3á6H2O in D2O. Necessary polyethylene vessels were used. measurements were carried out at room temperature after All lab-glassware were soaked in 10% HNO3 for at least 24 h, allowing the sample solution to stand for more than 24 h. then carefully washed with double-distilled water. The solution pH was adjusted by a buffer solution of NaAc with HAc (pH Cyclic voltammetry, UV-vis and Raman measurements 4.50), or by adding a sodium hydroxide solution. Cyclic voltammetry was performed using a BAS-100B Electrochemical Analyzer (USA) with a three-electrode system pH-metric measurements at 25ûC. A hanging mercury electrode (HMDE) was used as the The pH-metric titration was measured with a 77785 Titrator working electrode; a saturated calomel electrode (SCE) was Radiometer (Denmark) with a pair of electrodes (glass electrode used as a reference electrode and a platinum wire as an auxiliary and calomel electrode), which were first calibrated for the electrode. An inert gas, pure nitrogen, was used for degassing hydrogen-ion concentration according to Irving et al.22 The of solution prior to measurements. UV-vis spectra were proton and Al complexes stability constants were measured by recorded on a Shimadzu UV-3100-VIS-NIR spectrophotometer pH-metric titration of 50.0 ml samples. The temperature, (Japan); the splitting width was 0.5 nm. Raman spectra were 25±0.1ûC, was maintained by circulating thermostated water obtained on a Bruker RFS100 spectrophotometer (Swiss). through a jacket. Pure nitrogen gas was bubbled through the ANALYTICAL SCIENCES FEBRUARY 2003, VOL. 19 275

(a) (b)

Fig. 3 Species distribution curves in the (a) Cα-KG = 0.001 M, CAl = 0.001 M, I = 0.10 M KCl Al-α- KG system. (b) Cα-KG = 0.15 M, CAl = 0.05 M, I = 0.10 M KCl Al-α-KG system.

Results and Discussion

Complexation To assess the binding abilities of Al with α-ketoglutarate in aqueous solution, pH-metric studies were performed in an acidic aqueous solution. Alpha-ketoglutaric acid in its fully protonated form is designated as H2L. The carboxyl groups can be twice deprotonated.26 Three titrations were repeated for each sample solution of α-KG in the range of pH 3.02 Ð 10.61. The obtained pKa values are given in Table 1, which agree well with the reported values.26 When an equimolar quantity of Al3+ was added to a solution of α-KG (1 mM), the pH of the resulting solution decreased by approximately 1.02. For 1:1 Al-α-KG systems, the range of titration was limited from 2.0 to 5.3 Fig. 4 Cyclic voltammogram of Al-α-KG complexes in a three- because precipitation occurred above pH 5.3, and the solution electrode system; 1 mM α-KG, 0.10 M pH 4.50 HAcÐNaAc buffer became clear at pH 9.0, probably due to the low solubility of the solution; scan rate, 100 mV/s; L/Al ratio a, 1:0; b, 1:1/4; c, 1:1/2; d, metal complexes, or metastable Al-hydroxo species under this 1:1. condition. The deviation of the titration curve of Al-α-KG from that of α-KG, however, was sufficient to determine the complexes formed in the pH range of titration. Figure 2 shows an example of the titration curves of the pH versus the volume behaviors of α-ketoglutarate in both the presence and absence of the titrant for the ligand-to-metal (L/Al) ratios of 1:1, 2:1 and of Al using a cyclic voltammetry technique. Figure 4 shows a 5:1. To simulate the titration curve, 36 Ð 46 data points of each cyclic voltammogram of 1 mM α-ketoglutarate in the presence titration were used. of different L/Al ratios of 1 mM Al with a 0.10 M pH 4.50 The results of potentiometric data of the complexation HAcÐNaAc buffer solution. At a scan rate (v) of 100 mV/s, the equilibriums between α-KG and Al are given in Table 1. Also, major feature for the free ligand (curve a) was an irreversible

Fig. 3 (a) shows the distribution diagrams from pH 2.0 to pH process with a peak potential (EpC) of Ð1.18 V vs. SCE. 5.5 of Al and α-ketoglutarate at a concentration of 1 mM in the The possible adsorption redox process is assigned to the case of the Al:L = 1:1 system. At pH 4.5, except for Al3+, the reaction >C=O → >CHÐOH. Curves b to d give + 1:1 species AlL and AlLHÐ1 are predominant in acidic solution. voltammograms corresponding to different ratios of L/Al. In These two 1:1 complexes may be expected in equal amounts at the presence of Al, the free ligand peak decreased and a new 2+ about pH 4.7. Below pH 4.5, the mononuclear AlHL and irreversible redox peak appeared near Epc = Ð0.82 V vs. SCE. 4+ dinuclear Al2L species are increased, but are still small. The Those peaks can probably be assigned to the adsorption of the Ð 3Ð proportion of the 1:2 species AlL2 , AlL2HÐ2 increases with complexed ligand. A good linear relationship between the scan Ð1 increasing the L/Al ratio and the sample concentration. Figure rate (v/mV s ) and the peak current (ip/A) is observed (Fig. 5a), Ð 3Ð 3 (b) shows the 1:2 species AlL2 at pH 4.5 and AlL2HÐ2 at pH which means that the electroactive process is an irreversible 29 5.7, becoming the predominant enolic species in the CAl = 0.05 adsorption one. By using the equation 1/ip = 1/ip,max + 3Ð n M, Cα-KG = 0.15 M system. However, the 1:3 complex AlL3 is 1/βip,maxá1/CA (where ip is the measured peak current, ip,max is the 3+ neglected due to the small metal ion Al ; also, the ligand ionic peak current when all metal ions form the complex and CA is the sphere causes a steric inhibition. The computer calculation concentration of the ligand, n is the coordination number, β the n program also rejects it. Like glutamic acid, the tridentate conditional stability constant), a plot of 1/ip vs. 1/CA is shown coordinating ability of α-KG is much weaker owing to the in Fig. 5 (b). A straight line (n = 1) is obtained (r = 0.999), lower stability of the second seven-member chelating ring.27 which indicates that the composition (1:1) of the electroactive In order to understand the complexation of Al with α-KG in complexes on the surface of the mercury electrode is the same acidic aqueous solution, we compared the electrochemical as the complexes in the solution. It also agreed with a study of 276 ANALYTICAL SCIENCES FEBRUARY 2003, VOL. 19

(a)

Fig. 6 27Al-NMR spectra of α-KG (A) and Al-α-KG (B), at pD = 2.0, C = 0.05 M; Cα = 0.15 M. (b) Al -KG

(a)

Fig. 5 (a) Cyclic voltammogram of iP (A) vs. scan rate (mV/s): a, (b) 20; b, 40; c, 60; d, 80; e, 100; f, 140; g, 180; h, 220 in a three- electrode system; 1 mM α-KG and Al3+, 0.10 M pH 4.50 HAcÐNaAc buffer solution; and the relationship of the peak currents of CVs and n the scan rates. (b) The curve of the 1/iP dependence on the 1/C A (n = 1,2,3); CAl = 1 mM, 0.10 M pH 4.50 HAcÐNaAc buffer solution; scan range, Ð400 Ð Ð1400 mV; scan rate, 100 mV/s.

the previous potentiomeric titration. Al has a predominant tendency to form chelates with ligands containing oxygen donor sites, leading to the formation of five- or six-membered chelating rings. The values of the chemical shifts are much lower (0 Ð 5 ppm) for chelates containing six- 32,33 α membered octahedral chelate rings. With the α-KG, which Fig. 7 UV-vis spectra of Al- -ketoglutarate system, (a) Cα-KG = 1 is a typical carbonyl carboxylic acid, Al could form 1:1, 1:2 and mM, at pH 4.50 aqueous solution. L/Al ratio: a, 1:0; b, 1:1/4; c, 2:1 six-membered octahedral chelate at carbonyl and adjacent 1:1/2; d, 1:1; e, 1:2. (b) Cα-KG =1 mM, at 0.1 M pH = 4.50 carboxylic moieties, and the hydrolytic reaction of Al becomes HAcÐNaAc buffer solution, L/Al ratio: a, 1:0; b, 1:1/4; c, 1:1/2; d, 1:1; e, 1:2; f, 1:4. appreciable above pH 4.0.32 As shown in Fig. 6 (B), one peak at δ = 0.29 ppm in the presence of Al (0.05 M) and α-KG acid (0.15 M) at pD = 2.0 solution, therefore, should be contributed to the Al-α-KG complexes resonance. However, in other acidic with the increases of Al and the ligand concentration (>10 mM) pH regions, some resonances are undetectably broad, which and the L/Al ratios. It can be assumed that the formation 2+ 2+ may due to the Al-α-KG complexes asymmetric structures or proceeds according to the reaction Al(OH) + H2L ←→ AlHL because of using relatively low-performance magnets. + H2O. The binary complex may be found according to the 2+ Ð + According to studies of pH-metric titration, cyclic reaction Al(OH) + HL ←→ AlL + H2O. Upon increasing the voltammetry and 27Al-NMR spectra, as a “hard acid”, Al could pH, the following reaction may occur: Al(OH)2+ + L2Ð ←→ effectively coordinate with the carboxylate and carbonyl groups AlLHÐ1 + H2O. The second molecular of α-KG may be directly + + 2Ð 3Ð + of α-ketoglutaric acid in aqueous solution owing to the high bound to AlL : AlL + L ←→ AlL2HÐ2 + 2H , or it may react affinity of Al toward oxygen. The 1:1 chelating complexes are with mixed hydrolytic complexes to produce the biscomplex: Ð Ð the major forms in lower concentration solutions (1 mM). Al(OH)L + HL ←→ AlL2 + H2O. Aluminate and Al(OH)3 are However, the 2:1 (L/Al ratio) complex species are increased the dominating complexes at high pH values. ANALYTICAL SCIENCES FEBRUARY 2003, VOL. 19 277

Fig. 9 Raman spectra of α-KG (A) and Al-α-KG (B), at pH 5.2, CAl = 0.05 M; Cα-KG = 0.15 M.

signal (Fig. 1, i′) and the δ = 4.90 ppm suppose to the H signal (Fig. 1, h′) of enol-form of Al-α-KG. The peak δ = 4.84 ppm corresponds to the exchanged HDO.28 These spectra probably contributed to the formation of an enol-form complex in the Fig. 8 1H-NMR spectra of α-KG (A) and Al-α-KG (B), at pD 3.5, system. Therefore, it proved that the enolic form of complex C = 0.01 M; Cα = 0.05 M. Al -KG increases because Al promoted enolÐketo tautomerization. In order to further confirm the tautomerism phenomena of α- ketoglutarate with Al, we employed Raman-spectra techniques Tautomerization to examine α-KG and its Al complexes. The Raman spectra

UV-vis spectrometry is a common tool for structural analysis, were obtained to study the CAl = 0.05 M, Cα-KG = 0.15 M especially for the olefinic compound. As shown in Fig. 7 (a), in complexes system. Figure 9 shows the Raman spectra of α-KG the presence of a concentration of 1 mM α-ketoglutarate with with and without Al for pH 5.2 aqueous solutions. The most Al at different Al/L ratios, pH 4.50 aqueous solution (the significant stokes line (A) at about 1720.0 cmÐ1 and 1404.2 cmÐ1 ν solution pH was carefully adjusted by adding a 0.1 M sodium are ascribed to >C=O asymmetric stretching vibrations ( asCOOÐ) ν α hydroxide solution), the appearance of a declining absorption and symmetric stretching vibration ( sCOOÐ) of -KG, Ð1 band at 200 Ð 240 nm (λmax = 212 nm) upon adding Al ions to α- respectively, whereas ∆(νasÐνs) = 315.8 cm . In the presence of ketoglutarate occurred in the UV-vis spectra. The decrease in Al, the species-shifts (B) were obviously move to 1641.0 cmÐ1 Ð Ð1 Ð1 the intensities of ÐCOO band as a function of L/Al ratios may and 1414.6 cm , and ∆(νasÐνs) decreased to 226.4 cm . This contribute to the increased complexation of Al with the α- not only means the complexation of α-KG with Al,28 but is also ketoglutarate carboxyl moiety and the occurrence of π→π* attributed to the >C=C< vibration band (1641.0 cmÐ1), which is electron energy transitions. Meanwhile, the increased shoulder- verified by the enolic structure of the complexes. absorption band around 260 nm (240 Ð 280 nm) suggests the The ketoÐenol tautomerization of the Al-α-KG system has formation of an enolic chelating compound (unsaturated enolic been observed on UV-vis, 1H-NMR and Raman spectra. acid).17 It also indicates the occurrence of the tautomerization Concerning the structures of the complexes, we believe that of Al-α-ketoglutarate complexes. With an increase of the Al/L there exists equilibrium between the two forms: keto-form ratios, the absorption peaks increase, which elucidate the enol-form. The equilibrium is, however, shifted to the increasing amount of enolic structure compounds. In other left or right, depending on the different solvent, solution acidity words, Al can promote the enolization of α-ketoglutarate by and the concentration of Al-α-KG. At a lower concentration (1 chelating with the substrate. The lower absorption peak of the mM), the keto-form species is the major form (Fig. 3 a, AlL+), spectra shown around 330 nm (300 Ð 340 nm) suggests the because the keto-structure of α-ketoglutaric acid in dilute characteristic spectra of α-ketoacids. This very weak aqueous solution (<1 mM) is the dominating form in which the absorption is due to the carbonyl group n→π* weak electron carbonyl double bond is not broken.31 However, with an energy transitions.17,28 Also, these decreases of the absorption increase of the complex solution pH and concentration, the 3Ð peak contribute to the complexation of >C=O with Al. enol-form species is increased (Fig. 3b, AlL2HÐ2 and AlLHÐ1). Meanwhile, by using 0.1 M pH = 4.50 HAcÐNaAc as the Furthermore, comparing the thermodynamical properties, the reaction system buffer solution, as shown in Fig. 7 (b), it is also affinity of Al with the enol-form hydroxyl group is stronger clear that Al could promote α-KG tautomerization, which is than that of the keto-form carbonyl group, especially at high substantially increased at the λ = 260 nm shoulder-absorption concentration and pH solutions. In other words, the hydroxyl band. group in enol-form is a “hard base”; in contrast, the carbonyl To shed light on this interaction, we have employed the 1H- group in the keto-form is a “soft base”.30 NMR technique to examine the ligand and complexes. The 1H- NMR spectra of α-ketoglutarate acid and its Al complexes at Transamination the concentration of CAl = 10 mM; Cα-KG = 50 mM and pD = 3.5 Glutamate can be produced from α-ketoglutarate via a D2O solutions are shown in Fig. 8, The spectrum of ligand (A) transamination reaction catalyzed by glutamate dehydrogenase was found to consist of three clearly separated sets of signals: δ (GDH) or through other pathways. This reaction occurs in all = 2.16 ppm and δ = 2.49 ppm are the resonances in the keto- life forms. The GDH activity is allosterically regulated and can 1 form of α-KG two H (CH2) signals, while δ = 2.72 ppm is the catalyze the conversion of glutamate to α-ketoglutarate. The 14 resonance in the enol-form of α-KG H (CH2) signal. For the inhibitory effect of the Al ion can decrease the GDH activity. Al-α-KG complex spectrum (B), δ = 2.43 ppm suggests the H A possible explanation is that Al may react with the substrates 278 ANALYTICAL SCIENCES FEBRUARY 2003, VOL. 19

Conclusions

In this paper, the interactions of aluminum with α-ketoglutarate in aqueous solution were studied by means of many analytical techniques. It was proved that the 1:1 chelated complexes were formed in a dilute acidic solution. With an increase of the sample concentration and the L/Al ratios, the 2:1 (L/Al) species was also found to exist. It was found that aluminum not only binds α-ketoglutarate in a bidentate manner at the carboxylate and carbonyl moieties, but also promotes the substrate to tautomerization. Furthermore, according to the electrochemical behaviors, α-KG is supposed to act as an available low- molecular-mass ligand for the electrochemical determination of aluminum in biological system. Since Al has a 3+ oxidation state in biological systems, and Al primarily complexes with Fig. 10 Species distribution curves of Glu27 and α-KG complexes biocomponents through the electrostatic interaction, these with Al at the concentration: CAl = 1 mM, CGlu = Cα-KG = 1 mM, results may help us to understand the origin of the toxicity of neutral forms of Glu and α-KG are H2A and H2L, respectively. aluminum at a molecular level.

Acknowledgements α-ketoglutarate and glutamate. However, it is unclear how Al induces a conformational change where Glu or NAD has an This project is supported by National Science Foundation of effective affinity toward the enzyme. According to the China (No. 20075011, 49831005 and 29777013), Research potentiomeric titration analysis (Fig. 10), the Al ion has a higher Funding of the State Key Laboratory of Coordination Chemistry affinity for αÐketoglutarate than glutamate at the same of China, Nanjing University, and a Visiting Fellowship from concentration in terms of the electrostatic repulsive effect of the the Berkeley Lawrence National Laboratory of the USA. + 3+ 14 ÐNH3 group and the coordinating Al ion. It has been found that the strength of complexation to the Al ion strongly decreased in the sequence24 hydroxyl References carboxylic acid > carboxylic acid amino acid. Al has a strong affinity for citrate and isocitrate, which can explain the 1. J. F. Ma, S. J. Zheng, H. Matsumoto, and H. Hiradate, observed reduction in the ability of aconitase and isocitrate Nature, 1997, 390, 569. dehydrogenase to catalyze isocitrate and α-ketoglutarate 2. J. M. de laFuente, V. Ramirez-Romisez, J. L. Cabrera- production in the presence of the metal ion.14 However, this Ponce, and L. Herrersa-Estrella, Science, 1997, 276, 1566. simple explanation may not be suitable for Al with the α- 3. T. Yokoyama, H. Abe, T. Kurisaki, and H. Wakita, Anal. ketoglutarate and glutamate system due to the weak Sci., 1999, 15, 969. complexation. It is necessary to consider the enzyme GDH and 4. T. Yokoyama, T. Murata, S. Kinoshita, and H. Wakita, the coenzyme NAD/NADH primary binding sites and Anal. Sci., 1998, 14, 629. secondary conformational changes in a supermolecular system. 5. T. Yokoyama, H. Abe, T. Kurisaki, and H. Wakita, Anal. Although the effects of Al on biological systems have been Sci., 1999, 15, 393. extensively described, direct information concerning the 6. F. P. Zhang, S. P. Bi, J. Liu, X. D. Yang, X. L. Wang, L. molecular basis of its effects on enzyme systems and cell Yang, T. Yu, Y. J. Chen, L. M. Dai, and T. M. Yang, Anal. culture is rather scant. Al influences (inhibits or activates) not Sci., 2002, 18, 293. only acid-base enzymes, but also some redox enzymes 7. S. P. Bi, X. D. Yang, F. P. Zhang, X. L. Wang, and G. W. (including dehydrogenase).11,13 Most of them are activated by Zou, Fresenius J. Anal. Chem., 2001, 370, 984. some metal ions (Mg2+, Ca2+, Zn2+). The effect of Al in these 8. J. F. Ma, P. R. Ryan, and E. Delhaize, Tre. Sci., 2001, enzymatic processes is generally the displacement of metal ions. 6, 273. Therefore, we compared the Al-α-KG complex formation 9. C. Exley and J. D. Birchall, J. Theor. Biol., 1992, 159, 83. constants with other related biological metal ions α-KG 10. D. A. Atwood and B. C. Yearwood, J. Organometal. complexes. The formation constants (log k1) for α-ketoglutarate Chem., 2000, 600, 186. Fe3+, Cu2+ complexes were reported to be 5.2 and 5.0, 11. C. Exley, “Aluminum and Alzheimer’s Disease. The 2+ respectively, while Al-α-KG was 1.92 (log k1: AlHL ) in this Science that Describes the Link”, 2001, Elsevier, 361. work; these values are significantly higher than the log k1 values 12. D. Voet and J. G. Voet (ed.), “Biochemistry”, 2nd ed., of 1.13, 1.14 and 1.29 reported for this with Zn2+, Sr2+ and Ca2+, 1995, John Wilely & Sons Inc., New York, 376. respectively.18Ð20 This can be attributed to the greater binding 13. A. Y. Louie and T. J. Meade, Chem. Rev., 1999, 99, 2711. ability of the transition metals (Fe3+, Cu2+) in comparison with 14. P. Zatta, E. Lain, and C. Cagnolini, Eur. J. Biochem., 2000, the main-group metals (Al3+ and alkaline earth metals). 267, 3049. Therefore, it can be concluded that the sequence of the metal 15. C. Exley, N. C. Price, and J. D. Birchall, J. Inorg. ions complex with α-KG activities is Fe3+, Cu2+ > Al3+, Ni2+ > Biochem., 1994, 54, 297. Ca2+, Mg2+, Sr2+ and Zn2+. 16. Z. X. Xu, L. Fox, S. Melethil, L. Winberg, and M. Badr, J. Pharm. Exp. Ther., 1990, 254, 301. 17. R. Steiberger and F. H. Westheimer, J. Am. Chem. Soc., 1951, 73, 429. ANALYTICAL SCIENCES FEBRUARY 2003, VOL. 19 279

18. H. Scheidegger, W. Felty, and D. L. Leussing, J. Am. Physis”, 80th ed., 1999, CRC Press, Boca Raton, New Chem. Soc., 1970, 92, 808. York, 8 Ð 49. 19. J. T. Smith and V. M. Doctor, J. Inorg. Nucl. Chem., 1975, 27. T. Kiss, L. Sõvagõ, L. Tõth, A. Lakatos, R. Bentani, A. 37, 775. Tapparo, G. Bombi, and R. B. Martin, J. Chem. Soc. 20. J. Schubert and A. Lindenbaum, J. Am. Chem. Soc., 1952, Dalton Trans., 1997, 1967. 74, 3529. 28. D. Whittaker (ed.), “Interpreting Organic Spectra”, 2000, 21. R. Hamel and V. P. Appanna, J. Inorg. Biochem., 2001, 87, Athenaeum Press Ltd., Gateshead, Tyne & Wear, 59. 1. 29. W. R. Jin, X. L. Jia, and J. M. Lu, Electroanalysis, 1995, 22. H. Irving, M. G. Miles, and L. D. Pettit, Anal. Chim. Acta, 10, 962. 1967, 38, 475. 30. Y . F. Zhao and G. H. Zhou (ed.), “Elemental Organic 23. A. E. Martell and R. J. Motekaitis (ed.), “Determination Chemistry”, 1998, Tsinghua Univ. Press, Beijing, 113. and Use of Stability Constants”, 1992, VCH Publishers 31. P. T. Djurdjevic and M. Jelikic-Stankov, J. Pharm. Biomed. Inc., New York, 37. Anal., 1999, 19, 501. 24. S. L. Simpson, S. Sjöberg, and K. J. Powell, J. Chem. Soc., 32. S. N. Mhatre, R. K. Iyer, and P. N. Moorthy, Magn. Reson. Dalton Trans., 1995, 1799. Chem., 1993, 31, 169. 25. A. B. Akbarov, Sh. A. Pulatova, G. M. Mirkamilova, and 33. J. W. Akitt, Prog. Nucl. Magn. Reson. Spectrosc., 1989, 21, D. Zh. Iiyasova, Uzb. Khim. Zh., 1987, 5, 16. 1. 26. D. R. Lide (ed.), “CRC Handbook of Chemistry and

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