J . Am. Chem. SOC.1983, 105, 602-609
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Multinuclear NMR Studies on Al(II1) Complexes of ATP and Related Compounds S. J. Karlik, G. A. Elgavish,’ and G . L. Eichhorn* Contribution from the National Institutes of Health, National Institute on Aging, Gerontology Research Center, Baltimore, Maryland 21 224. Received May 10, 1982 Abstract: The N M R spectra of AI(II1) complexes of ATP, using 27AI,31P,and ‘H nuclei, frequently exhibit slow-exchange
features and can be used directly to study the characteristics of the complexes. The resolution of the *’AI peaks at low pH makes possible affinity calculations that show, e.g., that 97% of the AI(II1) is bound to ATP at pH 2. AI(II1) complexation generally leads to upfield shifts of the 31Ppeaks of ATP, as well as AMP, ADP, NADP, GMP, GDP, dATP, GTP and ortho-, pyro-, and triphosphate; occasional downfield shifts are also noted. The ’H peaks of ATP are also generally upfield shifted. The combination of all the N M R results indicates that AI(II1) forms at least four complexes with ATP, that there is generally more than one complex present under any set of conditions, and that the equilibria between these complexes shift dramatically with pH. AI(II1) has no effect on the ’H spectrum of adenosine, but it has some very similar effects on the 31Pspectra of triphosphate and ATP. The following Al(II1)-ATP complexes have been identified, in the order of appearance with increasing pH: (1) AI(II1) bound top- and y-phosphates, with no effect on the bases, (2) AI(II1) bound to y-phosphate and N-7 @-phosphate binding not certain), (3) AI(II1) bound to p- and y-phosphates, and the bases stacked, and (4) AI(II1) bound only to y-phosphates. Rates of exchange between these complexes have been calculated; they are in the range characteristic of aluminum complexes. T h e interaction of aluminum with cellular components has recently aroused interest because A1 has been implicated in a number of toxic biological processes, among them the accumulation of Al(II1) in localized areas of brain in Alzheimer’s disease2 and the toxicity due to Al(II1) produced in the course of lengthy kidney dialysis treatment leading to dialysis dementia.3 In t h e latter ailment the toxic effects of Al(II1) have been thought to be due to the binding of Al(II1) to ATP and the consequent inhibition of reactions that require ATP p a r t i ~ i p a t i o n . ~For this reason the complexes formed between ATP and Al(II1) a r e of some significance. The reactions of aluminum in aqueous solution are complicated by the fact that Al(II1) exists in a variety of species, whose relative concentration depends on t h e degree of binding of OH- groups and therefore on the P H . ~ We have therefore studied Al(II1) binding to ATP as a function of pH, using 27Al,31P,and ‘H N M R . Similar studies were carried out with aluminum complexes of AMP, ADP, NADP, GMP, GDP, dATP, GTP, and ortho-, pyro-, and triphosphate;6 a comparison of t h e N M R spectra of these complexes with those of ATP has proved helpful in the determination of various aspects of the structures of the A1-ATP complexes. This work has demonstrated a rather extraordinary degree of binding of AI(II1) to ATP at the extremes of pH, unusually good resolution of the peaks of complexed and uncomplexed ligand due to slow exchange, and a complicated series of equilibria between a t least four complexes of Al(II1) and ATP. Many features of the structures of these complexes have been worked out. Most of the experiments have been conducted under conditions that promote the formation of 1:l Al-ligand complexes and not ~~
(1) Current address: Comprehensive Cancer Center, University of Alabama in Birmingham, Birmingham, AL 35294. (2) (a) Crapper, D. R.; Krishnan, S. S.; Dalton, A. J. Science (Washington, D.C.)1973, 180, 511. (b) Crapper, D. R.; Karlik, S. J.; DeBoni, U. Aging (N.Y.)1978, 7 , 471. (c) Crapper, D. R.; Quittkat, S.; Krishnan, S. S.; Dalton, A. J.; DeBoni, U. Acra Neuropathol. 1980, 50, 19. (d) Perl, D. P.; Brody, A. R. Science (Washington, D.C.) 1980, 208, 297. (3) (a) Alfrey, A. C.; LeGendre, G. R.; Kaehny, W. D. N . Engl. J . Med. 1976, 294, 184. (b) Arieff, A. I.; Cooper, J.; Armstrong, D.; Lazaromitz, V. C. Ann. fnfern.Med. 1979, 90, 741. (c) Kovalchik, M. T.; Kaehny, W. D.; Hegg, A. P.; Jackson, J. T.; Alfrey, A. C. J . Lab. Clin. Med. 1978, 92, 712. (4) (a) Womack, F. C.; Colowick, S. P. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 5080. (b) Solheim, L. P.; Fromm, H . J. Biochemistry 1980, 19, 6074. (c) Solheim, L. P.; Fromm, H. J. Anal. Biochem. 1980, 109, 266. (d) Viola, R. E.; Morrison, J. F.; Cleland, W. W. Biochemistry 1980, 19, 3131. (5) B a a , C. F.; Messmer, R. E. “The Hydrolysis of Cations”;Wiley: New York, 1976. (6) Abbreviations used: AMP, adenosine 5’- monophosphate; ADP, adenosine 5’-diphosphate; NADP, nicotinamide adenine dinucleotide 2’phosphate; GMP, guanosine 5’-monophosphate; GDP, guanosine 5’-diphosphate; dATP, deoxyadenosine 5’-triphosphate; GTP, guanosine 5-triphosphate; TP, triphosphate.
complexes that contain more ligand than AI(II1). In this way these complex equilibria are a t least simplified to the extent that complexes with stoichiometries other than 1:l a r e avoided. Some evidence from studies with excess ligand also indicates that the 1 :1 complexes a r e preferred anyway. The Cr(II1) and Co(II1) complexes of ATP have been studied extensively by Cleland et al.’ The structural features of these trivalent metal complexes can therefore be compared with those of the Al(II1) complexes. Experimental Section Materials. Adenosine 5’-triphosphate (ATP) was purchased from
Sigma Chemical and passed through pretreated Chelex resin (Bio-Rad). The AIC1, stock was prepared from 99.9995% AIC1,.6H20 (GallardSchlesinger). This high-quality aluminum salt was employed to minimize paramagnetic contamination; final concentration of paramagnetic ions was thus 6. Downfield shifts and line broadening with increasing pH may be attributed to hydroxide ion participation in the aluminum-ligand field in reactions that exhibit fast exchange. Because the aluminum nucleus is quadrupolar, asymmetry in the ligand field produces an increase in the N M R line width. Thus, ligand substitution by a variable number of hydroxides can yield a variety of nonsymmetric aluminum environments with increased 27AlN M R line widths. This phenomenon has also been observed for complexes of aluminum with carboxyl ligands.* Although a pH-induced broadening suggests increased hydroxide participation, it is not possible with 27A1N M R to distinguish whether an increase in hydroxylation results in dissociation of the AI-ATP complex, because the ligand-field alteration may produce broadening whether or not phosphate continues to occupy coodination sites. Phosphorus-31 Spectra. A. AI and ATP. Analysis of the 3 ' P spectrum of A1-ATP complexes leads, first of all, to evidence that 1 :1 AI-ligand complexes predominate. The spectrum of an equimolar solution of A1C1, and ATP (10 mM each) a t pH 5 (Figure 2A) contains only two broad resonances. As the ATP concentration is increased beyond the 1 : l ratio of ATP/Al, relatively sharp resonance lines (-5-7-Hz width) are observed, superimposed on the two broad peaks (Figure 2, B-D). The chemical shifts of these sharp peaks are identical with those of ATP resonances in the absence of aluminum. The intensity of these free ATP resonances increases with increasing ATP/AI ratio while the broad resonances, which must be those for aluminumbound ATP, remain unchanged. We conclude that the exchange between bound and free ATP is also slow on the 31P N M R chemical shift time scale and that A1 tends to bind to only one molecule of ATP. With the exception of the experiment shown in Figure 2, all experimental solutions contain a 1:l ratio of AI to ATP. An examination of ,'Pspectra at various pHs (Figure 3) reveals that a number of complexes exist and that the equilibrium between these is greatly affected by pH, as may be expected. At pH 1, the spectrum consists of peaks at --12, -15, -21, -23, and --27 ppm (Figure 3A). The strong binding of aluminum to ATP indicated by the 27A1spectrum at pH as low as 1 (Figure IA) is confirmed by 31PN M R . The spectrum at pH 2 is similar to that a t pH 1, but the peak intensities are altered (Figure 3B). An
Karlik, Elgauish, and Eichhorn
604 J . Am. Chem. SOC.,Vol. 105, No. 3, 1983
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NMR spectra of 10 mM AICle and ATP at pH 1 (A) and increased to pH 2 (B), 4 ( C ) , 7 (D), 7.5 (E), 8 (F), and 12 (G), conditions as in Figure 1. See Discussion for assignments of T', (Y, P'', and p,l, resonances that appear in the presence of aluminum. Figure 3.
equimolar mixture of A1 and ATP is not soluble between pH -2.5 and 3.5. Four broad peaks are observed a t p H 4 (Figure 3C) because of the merger of two peaks observed a t lower pH. Resonances a t -19 and -25 ppm correspond to those also observed a t p H 1 and 2, although they are shifted downfield 2 ppm and exhibit a reduced intensity; these peaks are lost a t higher pH. The peaks a t -11 and -22 ppm (Figure 3C) remain relatively unshifted through further p H increases (Figure 3, D and E). Above p H 7, sharp resonances (5-7-Hz line width) appear in the AI-ATP spectra (Figure 3, E and F), corresponding to those of A T P in the absence of aluminum a t the same pH. The increase in uncomplexed A T P in the 31PN M R spectra reveals the onset of dissociation of the AI-ATP species. B. Aluminum and Other Phosphate Ligands. In order to be able to interpret the perturbations caused by A13+on the 31PN M R resonances of ATP, we examined the effect of aluminum on a number of other phosphate-containing compounds. These experiments were carried out at low pH with equimolar AI and ligand concentrations. In this p H range, the a- and @-phosphatesignals of diphosphates and the a and y signals of triphosphates are superimposed but, as we shall see, become separated on complexation. Binding of aluminum to orthophosphate broadens the 31PN M R signal and shifts it upfield 8.4, 12.4, and 17.6 ppm (Figure 4, A and B). The broadening and upfield shifts are observed when the aluminum is complexed with G M P and A M P and to the nonbridging phosphate of N A D P (Figure 4, G and H ) . When aluminum binds to a diphosphate, the resulting 31Psignals are also broadened and shifted upfield. The magnitude of the shift is 2.9 ppm for pyrophosphate and 2.8 and 3.5 ppm for A D P (Figure 4D) and 4.0, 7.6, and 14 ppm for the diphosphate bridge in N A D P (Figure 4, G and H). The effect of aluminum on triphosphate is still more complex. The 31Pspectrum of an equimolar mixture of aluminum with ATP (Figure 3A), G T P (Figure 4F), or triphosphate (TP) (Figure 5A) contains several broad peaks. One-half of the -11 ppm peak (Figure 4E), which corresponds to overlapping y- and a-phosphate resonances, is shifted upfield (Figure 4F); the magnitude of the shift is the same as that observed for diphosphates. Only the unshifted peak a t -1 1 ppm shows a net N O E when broad-band 'H decoupling is applied; the peak therefore must be due to a-phosphate. Thus, the y resonance is shifted upfield by aluminum, while the a resonance remains unshifted. Moreover, the
-10
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-20
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-30
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Figure 4. NMR spectra of A1 complexes with phosphate-containing ligands at pH 2: (A) orthophosphate; (B) orthophosphate and aluminum; (C) ADP; (D) ADP and Al; (E) GTP; (F) GTP and Al; (G) N A D P (H) NADP and AI. NMR conditions as in Figure 2; AI and ligand concentrations were 10 mM each.
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Figure 5. "P NMR spectra of 10 mM sodium triphosphate and 10 mM AIC13 mixed at pH 1 (A) and increased to pH 2 (B), 3 (C), 4 (D), 5 (E), and 6 (F). Conditions as in Figure 2.
@-phosphate a t -24 ppm (Figure 4E) is split into three peaks, including two additional resonances at -21 and -27 ppm (Figure 4F). The 31PN M R spectra of an equimolar mixture of aluminum and triphosphate (TP) were examined more closely, in order to observe any similarities and differences from those of A1-ATP mixtures (Figure 3), by obtaining a pH profile between pH 1 and 6 (Figure 5). At p H 1, the spectra are remarkably similar and consist of five resonances in nearly the same chemical shift positions. It is indeed striking that chemical shift alterations produced by aluminum on A T P and G T P a- and y-phosphates are very similar to those observed with TP. Although the a- and y-(terminal) phosphates of T P are identical, only one of these is perturbed by the binding of A1 between pH 1 and 3, in a manner identical with the behavior of ATP and GTP. Thus, binding to
NMR Studies on Al(III) Complexes of ATP
J . Am. Chem. SOC.,Vol. 105, No. 3, 1983 605
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chemical shifts of the phosphate groups for 10 m M ATP in the absence (a,j3,y) and in the presence (y', j3', b", 8"') of 10 mM A l a 3 . See Discussion for assignment of y', ,!?,' @",and B"' resonances. Figure 6.
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one phosphate in T P makes that phosphate resemble y-phosphate in ATP and renders the other terminus inactive, like a-phosphate in ATP. As the p H is increased, the peak a t -14 ppm (y-like) moves downfield and merges with the peak a t -10 ppm ( d i k e ) a t p H 4 (Figure 5B-D). This downfield shift probably reflects deprotonation of an aluminum-bound phosphate. The multiplicity of peaks observed for the center phosphate (corresponding to @) presumably reflects the formation of a number of complexes. As the p H approaches neutral, the TP spectrum again becomes virtually identical with the spectrum of ATP. Thus, the effect of Al(II1) on the 31P N M R spectrum of phosphate ligands depends primarily on the number of phosphates within the ligand and only to a minor degree on any other molecular component. C. Assignment of AI-ATP Spectra. Certain peak assignments of AI-ATP can be deduced by comparison with the model compounds. Thus the peaks upfield of the a-y region and the @ region can be ascribed to A1 bound to y- and @-phosphates,respectively. The assignment of the resonances of the AI-ATP complexes on the basis of these model studies was confirmed by measuring the peak areas of the 3'P N M R spectra of AI-ATP mixtures, starting a t high pH, because the resonances a t basic pH were readily identifiable as peaks from uncomplexed ATP. In the following discussion, Al-bound peaks are designated with one or more '. A t p H 12 (Figure 3G), the 31PN M R spectrum is nearly identical with A T P in the absence of aluminum, and therefore nearly all the ATP is dissociated from Al. At pH 8 (Figure 3E), the y and @ resonances are reduced and broad shoulders appear upfield of the a doublet and @ triplet. T h e a doublet does not appear to be affected. The sum of the areas of the y-phosphate doublet and the shoulder upfield of the a doublet (7') is equal to one phosphate, within experimental error, at pH
