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Metal contaminants in commercial preparations of nucleotides
ANALYTICAL
BIOCHEMISTRY
103, 87-93 (1980)
Metal Contaminants KEITH *Graduate Harold
TORNHEIM,*
Department E. Edgerton
in Commercial THOMAS
Preparations
R. GILBERT,?
of Biochemistry, Brandeis University, Research Laboratory, New England
of Nucleotidesl
AND JOHN M. LOWENSTEIN* Waltham, Aquarium,
Massachusetts 02254, Boston, Massachusetts
and tthe 02110
Received August 20, 1979 Commercial preparations of ATP contain aluminum in amounts as high as 0.3 mol%. Aluminum ion causes a change in the spectrum of the dye Eriochrome Blue, and the effect is potentiated by adenine nucleotides. Hexokinase is inhibited by aluminum; the inhibition is antagonized by magnesium and is slowly relieved by EDTA. Some commercial preparations of ATP and ADP contain nearly 1 mol% of magnesium. A commercial preparation of GTP contains 0.07 mol% copper and 1.4 mol% barium.
The dye Eriochrome Blue SE (3-[(5-chloro2 - hydroxyphenylkrzo] - 4,5 - dihydroxy - 2,7 naphthalenedisulfonic acid) has been used to measure the concentration of free magnesium ions (1,2). On adding ATP to a solution of the dye, we observed a spectral change in the absence of added magnesium ion. The spectral change was largely reversed by adding EDTA, which suggested the presence of a contaminating metal ion in the ATP. After adding ATP to the dye the full development of the spectral change took several minutes. Reversal of the spectral change by EDTA was slow by comparison with the fast changes observed when magnesium ion and subsequently EDTA were added to the dye in the presence or absence of ATP. Hence, the contaminating metal was not magnesium. Emission spectrometry revealed that aluminum is a major contaminant in some commercial preparations of ATP. The presence of this metal accounts for much of the spectral change observed when these preparations of ATP are added to the dye. Certain commercial preparations ’ This work was supported by National Institutes of Health Grant GM-07261. Publication 1293 from the Graduate Department of Biochemistry, Brandeis University, Waltham, Mass.
87
of ATP and ADP were found to contain relatively high amounts of magnesium. EXPERIMENTAL
PROCEDURE
Detection and measurement of aluminum, magnesium, barium, and copper by plasma emission spectrometry. The emission spec-
trometer used was a SpectraSpan III with a three-electrode plasma jet (SpectraMetrics, Inc.) with a Hamamatsu R292 photomultiplier. The operational parameters of the plasma jet were essentially those recommended by the manufacturer, except that the argon pressure to the pneumatic nebulizer was increased from 20 to 30 psi. The spectrometer entrance slit was 50 pm wide and 200 pm high. Qualitative detection of trace elements was made using a photographic attachment to the spectrometer to record the emission spectra. Quantitative determinations of aluminum, magnesium, and copper were made by measuring the intensity of emission at 394.403,279.553, and 327.396 nm, respectively. Barium was measured at 455.407 or 230.423 nm. Samples generally contained 50 mM nucleotide, except for 100 mM in the case of AMP, and about 200 mM sodium. Standard and blank solutions contained
OOO3-2697/8O/OSOO87-07$02.00/O Copyright 0 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.
88
TORNHEIM,
GILBERT,
200 mtvr NaCI. Standards were prepared by dilution of Fisher Certified Atomic Absorption Standards. Dye spectra. Difference spectra of Eriochrome Blue were run from 750 to 400 nm on a Perkin-Elmer model 557 recording spectrophotometer at 30°C. The reference cuvette contained 50 PM Eriochrome Blue, 50 mM triethanolamine-HCl buffer (25% neutralized with HCl, pH 8.3), and 150 mM NaCl. When present at a concentration of 10 mM, ATP contributed 40 mM sodium ion and 110 mM NaCl was added to maintain the total sodium ion concentration at 150 mM. Aluminum and other metals were added as the chlorides. The difference spectra were recorded after the development of the spectral change had gone to completion; this took up to 30 min when aluminum and ATP were present. Enzyme assays. Enzymatic reaction rates were measured in coupled assays at 30°C by following the absorbance change at 340 nm minus the absorbance change at 400 nm using a Perkin-Elmer model 356 two-wavelength spectrophotometer. The reaction mixture for hexokinase (EC 2.7.1.1) contained 50 mM triethanolamine-HCl buffer, pH 7.6,50 mM KCl, 1 mM glucose, 0.5 mg/ml NADP, 1 CLg/ml glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 0.1 &ml yeast hexokinase, and ATP and MgClz as indicated. The reaction mixture for pyruvate kinase (EC 2.7.1.40) at inhibitory levels of ATP contained 50 mM imidazole-HCl buffer, pH 7.0, 130 mM KCI, 0.06 mM phosphoenolpyruvate, 0.5 mM ADP, 0.25 mM NADH, 15 mM 0.5 mM orthophosphate, 1 mM M&l,, triethanolamine, 20 pg/ml lactate dehydrogenase (EC 1.1.1.27), 0.1 pg/ml pyruvate kinase, and 10 mM ATP which contributed 20 mM potassium ion and 20 mM sodium ion. The reaction mixtures for myokinase (EC 2.7.4.3), phosphofructokinase (EC 2.7.1.1 l), and creatine kinase (EC 2.7.3.2) contained 50 mM triethanolamine-HCl buffer, pH 7.6, 50 mM KCl, 0.5 mM phosphoenolpyruvate,
AND
LOWENSTEIN
0.25 mM NADH, 0.5 mM orthophosphate, 0.5 mM imidazole, 20 &ml lactate dehydrogenase, 20 pg/ml pyruvate kinase, and a concentration of MgCl, equal to 0.5 mM plus the ATP concentration. In addition, the reaction mixtures contained either 0.02 pg/ml myokinase, 0.5 mM AMP, and 0.251.0 mM ATP; or 1 pg/ml phosphofructokinase, 1 mM fructose 6-phosphate, and 0.1 or 0.2 mM ATP; or 0.1 pg/ml creatine kinase, 10 mM creatine, and 0.5 or 1.0 mM ATP. ATP from Boehringer Lot 1018349 was used in all enzyme assays. Aluminum was added from stock solutions containing ATP and 10 or 50 mol% of AlCl, as noted. Reactions were started by adding the enzyme to be tested. The reaction mixture for testing if aluminum could substitute for magnesium in the pyruvate kinase reaction contained 50 mM triethanolamine-HCl, pH 7.6, 150 mM KCl, 0.5 mM phosphoenolpyruvate, 5 mM ADP, 0.25 mM NADH, 20 pg/ml lactate dehydrogenase, 0. I &ml pyruvate kinase, and 0.1 to 5 mM AlCl,. Materials. Nucleotides were purchased from Sigma, Boehringer-Mannheim, and P-L Biochemicals. Eriochrome Blue SE and imidazole were obtained from Eastman, creatine from Sigma, NADP from P-L Biochemicals, and fructose 6-phosphate and NADH (Grade I) from Boehringer-Mannheim. Phosphoenolpyruvate (tricyclohexylammonium salt) was from Boehringer and Sigma. AlCl, was from Mallinckrodt. All other metal chlorides, glucose, and triethanolamine (certified grade) were from Fisher. All enzymes were obtained from Boehringer-Mannheim as suspensions in ammonium sulfate, except that creatine kinase was a lyophilized preparation. When lactate dehydrogenase and pyruvate kinase were used as coupling enzymes in the assays, they were freed of ammonium sulfate by gel filtration on a column of Sephadex G-25 equilibrated with 20 mM potassium phosphate, pH 7.0, or 20 mM imidazole-HCl, pH 7.0, respectively.
METAL
CONTAMINANTS
IN COMMERCIAL
RESULTS Aluminum and Magnesium in Commercial Preparations of Nucleotides
The emission spectra of some commercial preparations of ATP indicated the presence of substantial amounts of aluminum. Aluminum was then measured quantitatively from the emission intensity at 394.403 nm. Amounts of aluminum as high as 0.34 mol% were found in ATP isolated from yeast by Sigma and P-L, whereas Sigma ATP isolated from equine muscle and Boehringer ATP generally showed substantially lower levels of contamination by aluminum (Table 1). A few commercial preparations of other nucleotides were examined for comparison. Compared to the range found in ATP, low or very low levels of aluminum were found in
PREPARATIONS
OF NUCLEOTIDES
89
ADP, AMP, and GTP, with an intermediate amount in NAD. ATP from Boehringer and ADP from P-L contained nearly 1 mol% magnesium; all other nucleotide preparations tested had much lower levels. While the emphasis of this work was on aluminum and magnesium, we observed indications of barium and copper in some samples (Table 1). In particular, these metals are present in the GTP preparation at levels of 1.4 and 0.07 mol%, respectively. Interaction of Aluminum and ATP with Eriochrome Blue
At pH 8.3 Eriochrome Blue has a broad absorbance peak at 600 nm (band width about 150 nm). Addition of magnesium or
TABLE 1 ALUMINUM,MAGNESIUM,BARIUM,ANDCOPPERIN COMMERCIAL ATP ANDOTHERNUCLEOTIDES Amount (mmoYmol)* Nucleotide
Lot
Al
Mg
Ba
cu
1.3
0.02
<0.005
0.02
0.04
0.02
GTP
0.21 0.64 0.41 0.37 1.08 0.46 3.4 3.4 3.4 1.76 0.04 0.25 0.22 0.01 0.03 0.04 0.01 0.14
9.0 7.5 0.104
Boehringer Boehringer K-salt P-L Boehringer Sigma Sigma Free acid P-L Free acid P-L
1018349 1466239 48C-7150 38C-7170 32C-7440 12X-7250 96C-7140 96C-7170 61002 81011 1208549 1397101 76006 1366142 64C-7530 44C-7290 72003 620003
NAD
P-L
6303 1
0.79
ATP
Sourcea Boehringer Sigma
A-5394
Sigma
A-3 127
Sigma
A-2383
P-L ADP
AMP
0.31 0.151 0.116 0.154 0.011 1.83 7.5
14 0.05
0.7 0.1
a The commercial preparations are the sodium salts unless otherwise indicated. The three types of ATP from Sigma are: A-2383, from yeast; A-3127, from equine muscle; and A-5394, from equine muscle, vanadiumfree. AMP from Sigma was from yeast. NAD was the standard grade. b Blank spaces mean “not determined.”
90
TORNHEIM,
GILBERT,
other multivalent ions causes a shift of the absorbance peak to shorter wavelengths. The resulting difference spectrum has a trough in the 600 to 650 nm region and a peak below 600 nm. A similar spectral change is observed when 10 mM ATP is added to the dye in the absence of added divalent metal ions. The spectral change is large for the ATP preparations which are heavily contaminated with aluminum and is much smaller for ATP preparations which are low in aluminum. For example, difference spectra are shown in Fig. 1 for ATP from Boehringer Lot 1018349 (curve A) and Sigma Lot 96C-7140 (curve D). These two lots of ATP contributed 2 and 34 PM aluminum to the reaction mixture, respectively (Table 1). In the absence of ATP, addition of 34 PM Ah& causes a considerable shift in the spectrum of the dye (curve B), but the change is much smaller than that observed with the ATP from Sigma (curve D). If 34 PM AlC& and 10 mM ATP low in
O’I 0
-0.1 AA -02
-03 I 600
I
I 650
I
I 700
I 750
k inm)
FIG. 1. Difference spectra of Eriochrome Blue with: A, 10 mM ATP (Boehringer Lot 1018349); B, 34 PM AlC&; C, 10 mM ATP (Boehringer Lot 1018349) plus 34 pM AICI,; and D, 10 mM ATP (Sigma A-2383, Lot %C-7140). The reference solution lacked both ATP and aluminum. The solutions contained 50 PM Eriochrome Blue, 50 mM triethanolamine-HCl buffer (25% neutralized with HCl, pH 8.3), and 150 mM sodium ion from ATP and NaCI.
AND LOWENSTEIN
aluminum are added together (curve C), a much larger effect is observed than would be expected from the sum of the separate effects shown in curves A and B, and the resulting difference spectrum is much closer to that shown by the ATP preparation contaminated with a similar amount of aluminum (curve D). It is concluded that most of the spectral change seen when ATP is added to Eriochrome Blue is due to contamination of commercial preparations of ATP with aluminum. The change in spectrum of the dye caused by aluminum is potentiated by the presence of ATP, as is indicated by curves A, B, and C in Fig. 1. This suggests the formation of a ternary complex of dyealuminum- ATP. Calculations from difference spectra taken in the presence of 40 PM aluminum chloride and 0, 1, 2, 5, and 10 mM ATP (Boehringer Lot 1018349) gave a KD of 10 mM ATP for dissociation of the ternary complex, when l/[ATP] was plotted against 1/(AA6,,,, - AAd or against l/( AA,,,, - AA&. ADP is more effective than ATP at potentiating the spectral change of the dye caused by aluminum, while AMP is less effective. For example, if the absorbance change at 625 nm for 40 PM aluminum plus 10 mM nucleotide is compared with the sum of the changes for aluminum alone and nucleotide alone, then the difference in absorbance units is 0.09 for ATP (Boehringer Lot 1018349), 0.27 for ADP (Boehringer Lot 1208549), and 0.03 for AMP (P-L Lot 72003), respectively. The spectral changes produced by aluminum in the absence of ATP increase monotonically over the range 20 to 500 j.bM AQ, although the continuous shifting of the isobestic point indicates that more than just a single binary complex is formed. With Mg2+ and Ca2+ there is little if any shift of the isobestic point; dissociation constants of the dye-metal complexes are about 0.1 and 1 mM, respectively, under the conditions used here and the maximum
METAL
CONTAMINANTS
IN COMMERCIAL
absorbance change is about 0.5 unit. The affinity of the dye for Mg2+, and possibly for other metals, is less at lower pH. The dye has been used to determine the dissociation constant for the Mg-ATP complex over a range of magnesium concentrations (unpublished work). The same value of the dissociation constant is obtained in the presence of 0.5 or 10 mM ATP (Boehringer Lot 1018349), which indicates that the MgATP complex has little if any effect on the dye. ATP from Boehringer Lot 1018249 contains 0.9 mol% of magnesium (Table 1). This contaminant does not contribute significantly to the effect of ATP on the dye. The apparent dissociation constant for the Mg-ATP complex is about 60 FM under conditions used here (150 mM sodium ion and pH 8.3), which means that over 9% of the contaminating magnesium would be chelated at a concentration of 10 mM ATP and free magnesium ion would be less than 1 PM. The preparations of GTP and NAD which were tested also cause a spectral change of the dye when added at a concentration of 10 mM. The trough in the difference spectra is at about 620 nm, and the absorbance change is about 0.28. ADP (K-salt, Boehringer Lot 1397101) 8 mrvr, produces a trough at 615 nM, with an absorbance change of 0.33, 10 times that of the sodium salt (Boehringer Lot 1208549). In these three cases, too, the spectral changes take a period of time to develop fully and are but slowly reversed by addition of EDTA, indicating contamination by metal ions. The amounts of aluminum in these three preparations (Table 1) are too low to be responsible for much of the spectral change, with the possible exception of the NAD preparation, if this substance has as great a potentiating effect as ADP. Fe3+ Cu2+ MnZ+, and Ca2+ interact with the dye, but ‘no potentiation of the absorbance changes is observed on adding 10 mM ATP (Boehringer Lot 1018349) to 10 FM
PREPARATIONS
OF NUCLEOTIDES
91
Fe3+, Cu2+, or Mn2+, or 100 pM Ca*+. The spectrum of the dye exhibits biphasic behavior with increasing concentrations of Cu*+ in the absence of ATP. The absorbance change increases between 10 and 50 FM Cu*+ but decreases slightly between 50 and 100 j&M cLl*+ with a slight shift of the isobestic point. At 1 mM Cu*+ the absorbance trough has shifted from 622 to 636 nm, and the maximum absorbance difference is reduced by half, compared with 50 pM Cu*+. Inhibition
of Hexokinase
by Aluminum
The actual substrate for most ATPutilizing enzymes is the magnesium-ATP complex. The AP+ ion is likely to form a very tight complex with ATP, which might compete with Mg-ATP in some cases. Yeast hexokinase is inhibited by aluminum, and the inhibition is antagonized by raising the magnesium concentration. Thus, in the presence of 0.2 mM ATP (Boehringer Lot 1018349), 0.02 mM AlCl, causes 45% inhibition with 0.7 mM MgCl, (concentration of free Mg*+ = 0.5 mM) and 35% inhibition with 1.2 mM MgC12 (free Mg2+ = 1 mM). In the absence of aluminum the rate is unaffected by increasing the MgCl, concentration from 0.7 to 1.2 mM. If the aluminum concentration is increased to 0.1 mM, the inhibition is 72 and 56% in the presence of 0.7 and 1.2 mM MgCl,, respectively. However, part of this inhibition may simply be due to the aluminum tying up a portion of the ATP in an aluminum-ATP complex and thus reducing the amount of Mg-ATP available for use as substrate. If 0.1 mM aluminum binds an equal amount of ATP, then the substrate concentration would be reduced from 0.2 to 0.1 mM ATP. The possible contribution of this factor to the inhibition by aluminum can be assessed by comparing the rate in the presence of 0.1 mM AlCl, and 0.2 mM ATP with the rate of a control containing only 0.1 mM ATP. This sets a lower limit to the degree of direct inhibition by aluminum, or the Al-ATP
92
TORNHEIM,
GILBERT,
complex, of 61 and 40% in the presence of 0.7 and 1.2 mM MgC&, respectively. The steady-state rate in the presence of aluminum is about the same whether the reaction is started by adding hexokinase, or glucose, or magnesium. However, when the reaction is started by adding hexokinase or glucose, the rate in the presence of aluminum is faster initially and slows down to a constant rate over a period of l-2 min; when the reaction is started by adding magnesium, the rate is slower initially, and then speeds up to a constant rate over a period of about 2 min. In the absence of aluminum, the rate with 0.1 mM ATP is 71% of the rate with 0.2 mM ATP, consistent with a K, for ATP of about 0.2 mM (3). When 0.4 mM EDTA is added to a reaction mixture containing 0.2 mM ATP, 0.1 mM AlC&, and 1.2 mM MgC12, the rate first decreases to a level consistent with the reduction in the free magnesium concentration and then rises slowly. The gradual increase in rate presumably represents removal of the inhibitory aluminum, since addition of EDTA in the absence of aluminum has little effect. However, when 100 times the usual amount of hexokinase is added to a reaction mixture containing 0.2 mM ATP, 0.1 mM AlC&, and 0.7 mM MgClz, all the ATP is utilized within about 6 min. With 10 times the normal amount of hexokinase, 90% of the ATP is utilized in 40 min; the reaction proceeds steadily with no indication of a biphasic process which would reflect a slower utilization of a substantial portion of the ATP, as might be expected if dissociation of the AlATP complex becomes rate limiting. When present at a level equal to one-tenth the ATP concentration, aluminum does not inhibit myokinase, creatine kinase, or phosphofructokinase (at noninhibitory levels of ATP) by more than lo%, which is the degree of inhibition that might occur if the Al-ATP complex does not bind to the enzyme. The ATP concentrations were chosen to be near the K, values. The
AND
LOWENSTEIN
inhibition of pyruvate kinase by 10 mM ATP is also not affected significantly by addition of 1 mM A13+ and A13+ does not substitute for Mg2+ in the pyruvate kinase reaction. DISCUSSION
Metal binding dyes are useful for measuring the free concentrations of magnesium and calcium ions in biochemical systems. However, the results presented here add a cautionary note that the levels of metal contaminants present in commercial nucleotide preparations and, perhaps, in other biochemicals can interfere considerably with such measurements. This is especially true for aluminum ions, since chelation of such ions by adenine nucleotides increases rather than decreases the effect of the metal on the dye Eriochrome Blue (Fig. 1). On the other hand, a low specificity makes a dye useful for detecting the presence of metal contaminants which might influence enzymatic reactions. Hexokinase can be substantially inhibited by aluminum ions; however, note that the concentrations of aluminum ions used here were larger than the level of contamination found in commercial preparations of ATP. The antagonism of the aluminum inhibition by magnesium is consistent with the inhibitory form being the Al-ATP complex, but a direct effect of aluminum on the enzyme is not ruled out. In studies of the inhibition of several kinases by chromium-ATP, hexokinase was the most strongly inhibited, with a Ki of 0.35 pM; by comparison the Ki with creatine kinase was 140 pM (4). As observed here with aluminum, the hexokinase reaction in the presence of chromium-ATP slowed down over several minutes to a lower rate, which suggested tighter binding of the analog as a function of time (5). Aluminum is generally considered to be nontoxic, except when such large quantities are ingested that a substantial amount of phosphate is complexed and energy metabolism
METAL
CONTAMINANTS
IN COMMERCIAL
becomes affected (6). The normal blood level of aluminum is about 18 PM, whereas the toxic level is about 90 mM (7). Aluminum is found in many tissues (8,9) and may be of physiological import (10). Plants contain 10 to 30 mg aluminum/kg fresh weight. The daily dietary intake of aluminum is about 45 mg for adults in the United States (6). It is possible that some biochemical processes could be affected by moderate doses of aluminum without being easily detectable, ATP that is heavily contaminated with aluminum causes a greater spectral change of the dye than can be accounted for by the aluminum contamination alone, even considering the potentiating effect of ATP (Fig. 1). This suggests the presence of additional metal contaminants which, although present in smaller amounts than aluminum, could be more potent in their biochemical effects. Since rather high concentrations of ATP or ADP are required to potentiate the spectral change caused by aluminum, the possibility must be considered that the potentiation is due to some other contaminant in the adenine nucleotides. In view of the magnesium contamination found in some commercial preparations of ATP and ADP (Table 1), experiments purporting to show a lack of magnesium requirement for an ATP-utilizing reaction should include a determination of the presence or absence of magnesium in the particular nucleotide preparations used.
PREPARATIONS
OF NUCLEOTIDES
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Note added in proof. Womack and Colowick (1 I) have recently reported inhibition of hexokinase by aluminum present as a contaminant in some commercial preparations of ATP and ADP. The inhibition by aluminum is much stronger at a pH lower than that used in our studies.
REFERENCES 1. Scarpa, A. (1974) Biochemistry 13, 2789-2794. Scarpa, A. (1976) in Ion and Enzyme Electrodes in Biology and Medicine (Kessler, M., Clark, L. C., Jr., Lubbers, D. W., Silver, I. A., and Simon, W., eds.), pp. 252-260, University Park Press, Baltimore. 3. Fromm, H. J., and Zewe, V. (1962)5. Biol. Chem. 2.
237, 3027-3032.
4. Janson, C. A., and Cleland, W. W. (1974) .I. Biol. Chem. 249, 2572-2574. 5. Danenberg, K. D., and Cleland, W. W. (1975) Biochemistry 14, 28-39. 6. Venugopal, B., and Luckey, T. D. (1978) Metal Toxicity in Mammals, Vol. 2, pp. 105-113, Plenum, New York. 7. Williams, D. R. (1971) The Metals of Life, p. 56, Van Nostrand Reinhold, London. 8. Tipton, I. H. (1960) in Metal-Binding in Medicine (Seven, M. J., and Johnson, L. A., eds.), pp. 27-42, Lippincott, Philadelphia. 9. Butt, E. M., Nusbaum, R. E., Gilmore, T. C., and DiDio, S. L. (1960) in Metal-Binding in Medicine (Seven, M. J., and Johnson, L. A., eds.), pp. 43-49, Lippincott, Philadelphia. 10. Weinberg, E. D. (196O)in Metal-Binding in Medicine (Seven, M. J., and Johnson, L. A., eds.). pp. 324-334, Lippincott, Philadelphia. 11. Womack, F. C., and Colowick, S. P. (1979) Proc. Natl. Acad. Sci. USA 16. 5080-5084.
BIOCHEMISTRY
103, 87-93 (1980)
Metal Contaminants KEITH *Graduate Harold
TORNHEIM,*
Department E. Edgerton
in Commercial THOMAS
Preparations
R. GILBERT,?
of Biochemistry, Brandeis University, Research Laboratory, New England
of Nucleotidesl
AND JOHN M. LOWENSTEIN* Waltham, Aquarium,
Massachusetts 02254, Boston, Massachusetts
and tthe 02110
Received August 20, 1979 Commercial preparations of ATP contain aluminum in amounts as high as 0.3 mol%. Aluminum ion causes a change in the spectrum of the dye Eriochrome Blue, and the effect is potentiated by adenine nucleotides. Hexokinase is inhibited by aluminum; the inhibition is antagonized by magnesium and is slowly relieved by EDTA. Some commercial preparations of ATP and ADP contain nearly 1 mol% of magnesium. A commercial preparation of GTP contains 0.07 mol% copper and 1.4 mol% barium.
The dye Eriochrome Blue SE (3-[(5-chloro2 - hydroxyphenylkrzo] - 4,5 - dihydroxy - 2,7 naphthalenedisulfonic acid) has been used to measure the concentration of free magnesium ions (1,2). On adding ATP to a solution of the dye, we observed a spectral change in the absence of added magnesium ion. The spectral change was largely reversed by adding EDTA, which suggested the presence of a contaminating metal ion in the ATP. After adding ATP to the dye the full development of the spectral change took several minutes. Reversal of the spectral change by EDTA was slow by comparison with the fast changes observed when magnesium ion and subsequently EDTA were added to the dye in the presence or absence of ATP. Hence, the contaminating metal was not magnesium. Emission spectrometry revealed that aluminum is a major contaminant in some commercial preparations of ATP. The presence of this metal accounts for much of the spectral change observed when these preparations of ATP are added to the dye. Certain commercial preparations ’ This work was supported by National Institutes of Health Grant GM-07261. Publication 1293 from the Graduate Department of Biochemistry, Brandeis University, Waltham, Mass.
87
of ATP and ADP were found to contain relatively high amounts of magnesium. EXPERIMENTAL
PROCEDURE
Detection and measurement of aluminum, magnesium, barium, and copper by plasma emission spectrometry. The emission spec-
trometer used was a SpectraSpan III with a three-electrode plasma jet (SpectraMetrics, Inc.) with a Hamamatsu R292 photomultiplier. The operational parameters of the plasma jet were essentially those recommended by the manufacturer, except that the argon pressure to the pneumatic nebulizer was increased from 20 to 30 psi. The spectrometer entrance slit was 50 pm wide and 200 pm high. Qualitative detection of trace elements was made using a photographic attachment to the spectrometer to record the emission spectra. Quantitative determinations of aluminum, magnesium, and copper were made by measuring the intensity of emission at 394.403,279.553, and 327.396 nm, respectively. Barium was measured at 455.407 or 230.423 nm. Samples generally contained 50 mM nucleotide, except for 100 mM in the case of AMP, and about 200 mM sodium. Standard and blank solutions contained
OOO3-2697/8O/OSOO87-07$02.00/O Copyright 0 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.
88
TORNHEIM,
GILBERT,
200 mtvr NaCI. Standards were prepared by dilution of Fisher Certified Atomic Absorption Standards. Dye spectra. Difference spectra of Eriochrome Blue were run from 750 to 400 nm on a Perkin-Elmer model 557 recording spectrophotometer at 30°C. The reference cuvette contained 50 PM Eriochrome Blue, 50 mM triethanolamine-HCl buffer (25% neutralized with HCl, pH 8.3), and 150 mM NaCl. When present at a concentration of 10 mM, ATP contributed 40 mM sodium ion and 110 mM NaCl was added to maintain the total sodium ion concentration at 150 mM. Aluminum and other metals were added as the chlorides. The difference spectra were recorded after the development of the spectral change had gone to completion; this took up to 30 min when aluminum and ATP were present. Enzyme assays. Enzymatic reaction rates were measured in coupled assays at 30°C by following the absorbance change at 340 nm minus the absorbance change at 400 nm using a Perkin-Elmer model 356 two-wavelength spectrophotometer. The reaction mixture for hexokinase (EC 2.7.1.1) contained 50 mM triethanolamine-HCl buffer, pH 7.6,50 mM KCl, 1 mM glucose, 0.5 mg/ml NADP, 1 CLg/ml glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 0.1 &ml yeast hexokinase, and ATP and MgClz as indicated. The reaction mixture for pyruvate kinase (EC 2.7.1.40) at inhibitory levels of ATP contained 50 mM imidazole-HCl buffer, pH 7.0, 130 mM KCI, 0.06 mM phosphoenolpyruvate, 0.5 mM ADP, 0.25 mM NADH, 15 mM 0.5 mM orthophosphate, 1 mM M&l,, triethanolamine, 20 pg/ml lactate dehydrogenase (EC 1.1.1.27), 0.1 pg/ml pyruvate kinase, and 10 mM ATP which contributed 20 mM potassium ion and 20 mM sodium ion. The reaction mixtures for myokinase (EC 2.7.4.3), phosphofructokinase (EC 2.7.1.1 l), and creatine kinase (EC 2.7.3.2) contained 50 mM triethanolamine-HCl buffer, pH 7.6, 50 mM KCl, 0.5 mM phosphoenolpyruvate,
AND
LOWENSTEIN
0.25 mM NADH, 0.5 mM orthophosphate, 0.5 mM imidazole, 20 &ml lactate dehydrogenase, 20 pg/ml pyruvate kinase, and a concentration of MgCl, equal to 0.5 mM plus the ATP concentration. In addition, the reaction mixtures contained either 0.02 pg/ml myokinase, 0.5 mM AMP, and 0.251.0 mM ATP; or 1 pg/ml phosphofructokinase, 1 mM fructose 6-phosphate, and 0.1 or 0.2 mM ATP; or 0.1 pg/ml creatine kinase, 10 mM creatine, and 0.5 or 1.0 mM ATP. ATP from Boehringer Lot 1018349 was used in all enzyme assays. Aluminum was added from stock solutions containing ATP and 10 or 50 mol% of AlCl, as noted. Reactions were started by adding the enzyme to be tested. The reaction mixture for testing if aluminum could substitute for magnesium in the pyruvate kinase reaction contained 50 mM triethanolamine-HCl, pH 7.6, 150 mM KCl, 0.5 mM phosphoenolpyruvate, 5 mM ADP, 0.25 mM NADH, 20 pg/ml lactate dehydrogenase, 0. I &ml pyruvate kinase, and 0.1 to 5 mM AlCl,. Materials. Nucleotides were purchased from Sigma, Boehringer-Mannheim, and P-L Biochemicals. Eriochrome Blue SE and imidazole were obtained from Eastman, creatine from Sigma, NADP from P-L Biochemicals, and fructose 6-phosphate and NADH (Grade I) from Boehringer-Mannheim. Phosphoenolpyruvate (tricyclohexylammonium salt) was from Boehringer and Sigma. AlCl, was from Mallinckrodt. All other metal chlorides, glucose, and triethanolamine (certified grade) were from Fisher. All enzymes were obtained from Boehringer-Mannheim as suspensions in ammonium sulfate, except that creatine kinase was a lyophilized preparation. When lactate dehydrogenase and pyruvate kinase were used as coupling enzymes in the assays, they were freed of ammonium sulfate by gel filtration on a column of Sephadex G-25 equilibrated with 20 mM potassium phosphate, pH 7.0, or 20 mM imidazole-HCl, pH 7.0, respectively.
METAL
CONTAMINANTS
IN COMMERCIAL
RESULTS Aluminum and Magnesium in Commercial Preparations of Nucleotides
The emission spectra of some commercial preparations of ATP indicated the presence of substantial amounts of aluminum. Aluminum was then measured quantitatively from the emission intensity at 394.403 nm. Amounts of aluminum as high as 0.34 mol% were found in ATP isolated from yeast by Sigma and P-L, whereas Sigma ATP isolated from equine muscle and Boehringer ATP generally showed substantially lower levels of contamination by aluminum (Table 1). A few commercial preparations of other nucleotides were examined for comparison. Compared to the range found in ATP, low or very low levels of aluminum were found in
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ADP, AMP, and GTP, with an intermediate amount in NAD. ATP from Boehringer and ADP from P-L contained nearly 1 mol% magnesium; all other nucleotide preparations tested had much lower levels. While the emphasis of this work was on aluminum and magnesium, we observed indications of barium and copper in some samples (Table 1). In particular, these metals are present in the GTP preparation at levels of 1.4 and 0.07 mol%, respectively. Interaction of Aluminum and ATP with Eriochrome Blue
At pH 8.3 Eriochrome Blue has a broad absorbance peak at 600 nm (band width about 150 nm). Addition of magnesium or
TABLE 1 ALUMINUM,MAGNESIUM,BARIUM,ANDCOPPERIN COMMERCIAL ATP ANDOTHERNUCLEOTIDES Amount (mmoYmol)* Nucleotide
Lot
Al
Mg
Ba
cu
1.3
0.02
<0.005
0.02
0.04
0.02
GTP
0.21 0.64 0.41 0.37 1.08 0.46 3.4 3.4 3.4 1.76 0.04 0.25 0.22 0.01 0.03 0.04 0.01 0.14
9.0 7.5 0.104
Boehringer Boehringer K-salt P-L Boehringer Sigma Sigma Free acid P-L Free acid P-L
1018349 1466239 48C-7150 38C-7170 32C-7440 12X-7250 96C-7140 96C-7170 61002 81011 1208549 1397101 76006 1366142 64C-7530 44C-7290 72003 620003
NAD
P-L
6303 1
0.79
ATP
Sourcea Boehringer Sigma
A-5394
Sigma
A-3 127
Sigma
A-2383
P-L ADP
AMP
0.31 0.151 0.116 0.154 0.011 1.83 7.5
14 0.05
0.7 0.1
a The commercial preparations are the sodium salts unless otherwise indicated. The three types of ATP from Sigma are: A-2383, from yeast; A-3127, from equine muscle; and A-5394, from equine muscle, vanadiumfree. AMP from Sigma was from yeast. NAD was the standard grade. b Blank spaces mean “not determined.”
90
TORNHEIM,
GILBERT,
other multivalent ions causes a shift of the absorbance peak to shorter wavelengths. The resulting difference spectrum has a trough in the 600 to 650 nm region and a peak below 600 nm. A similar spectral change is observed when 10 mM ATP is added to the dye in the absence of added divalent metal ions. The spectral change is large for the ATP preparations which are heavily contaminated with aluminum and is much smaller for ATP preparations which are low in aluminum. For example, difference spectra are shown in Fig. 1 for ATP from Boehringer Lot 1018349 (curve A) and Sigma Lot 96C-7140 (curve D). These two lots of ATP contributed 2 and 34 PM aluminum to the reaction mixture, respectively (Table 1). In the absence of ATP, addition of 34 PM Ah& causes a considerable shift in the spectrum of the dye (curve B), but the change is much smaller than that observed with the ATP from Sigma (curve D). If 34 PM AlC& and 10 mM ATP low in
O’I 0
-0.1 AA -02
-03 I 600
I
I 650
I
I 700
I 750
k inm)
FIG. 1. Difference spectra of Eriochrome Blue with: A, 10 mM ATP (Boehringer Lot 1018349); B, 34 PM AlC&; C, 10 mM ATP (Boehringer Lot 1018349) plus 34 pM AICI,; and D, 10 mM ATP (Sigma A-2383, Lot %C-7140). The reference solution lacked both ATP and aluminum. The solutions contained 50 PM Eriochrome Blue, 50 mM triethanolamine-HCl buffer (25% neutralized with HCl, pH 8.3), and 150 mM sodium ion from ATP and NaCI.
AND LOWENSTEIN
aluminum are added together (curve C), a much larger effect is observed than would be expected from the sum of the separate effects shown in curves A and B, and the resulting difference spectrum is much closer to that shown by the ATP preparation contaminated with a similar amount of aluminum (curve D). It is concluded that most of the spectral change seen when ATP is added to Eriochrome Blue is due to contamination of commercial preparations of ATP with aluminum. The change in spectrum of the dye caused by aluminum is potentiated by the presence of ATP, as is indicated by curves A, B, and C in Fig. 1. This suggests the formation of a ternary complex of dyealuminum- ATP. Calculations from difference spectra taken in the presence of 40 PM aluminum chloride and 0, 1, 2, 5, and 10 mM ATP (Boehringer Lot 1018349) gave a KD of 10 mM ATP for dissociation of the ternary complex, when l/[ATP] was plotted against 1/(AA6,,,, - AAd or against l/( AA,,,, - AA&. ADP is more effective than ATP at potentiating the spectral change of the dye caused by aluminum, while AMP is less effective. For example, if the absorbance change at 625 nm for 40 PM aluminum plus 10 mM nucleotide is compared with the sum of the changes for aluminum alone and nucleotide alone, then the difference in absorbance units is 0.09 for ATP (Boehringer Lot 1018349), 0.27 for ADP (Boehringer Lot 1208549), and 0.03 for AMP (P-L Lot 72003), respectively. The spectral changes produced by aluminum in the absence of ATP increase monotonically over the range 20 to 500 j.bM AQ, although the continuous shifting of the isobestic point indicates that more than just a single binary complex is formed. With Mg2+ and Ca2+ there is little if any shift of the isobestic point; dissociation constants of the dye-metal complexes are about 0.1 and 1 mM, respectively, under the conditions used here and the maximum
METAL
CONTAMINANTS
IN COMMERCIAL
absorbance change is about 0.5 unit. The affinity of the dye for Mg2+, and possibly for other metals, is less at lower pH. The dye has been used to determine the dissociation constant for the Mg-ATP complex over a range of magnesium concentrations (unpublished work). The same value of the dissociation constant is obtained in the presence of 0.5 or 10 mM ATP (Boehringer Lot 1018349), which indicates that the MgATP complex has little if any effect on the dye. ATP from Boehringer Lot 1018249 contains 0.9 mol% of magnesium (Table 1). This contaminant does not contribute significantly to the effect of ATP on the dye. The apparent dissociation constant for the Mg-ATP complex is about 60 FM under conditions used here (150 mM sodium ion and pH 8.3), which means that over 9% of the contaminating magnesium would be chelated at a concentration of 10 mM ATP and free magnesium ion would be less than 1 PM. The preparations of GTP and NAD which were tested also cause a spectral change of the dye when added at a concentration of 10 mM. The trough in the difference spectra is at about 620 nm, and the absorbance change is about 0.28. ADP (K-salt, Boehringer Lot 1397101) 8 mrvr, produces a trough at 615 nM, with an absorbance change of 0.33, 10 times that of the sodium salt (Boehringer Lot 1208549). In these three cases, too, the spectral changes take a period of time to develop fully and are but slowly reversed by addition of EDTA, indicating contamination by metal ions. The amounts of aluminum in these three preparations (Table 1) are too low to be responsible for much of the spectral change, with the possible exception of the NAD preparation, if this substance has as great a potentiating effect as ADP. Fe3+ Cu2+ MnZ+, and Ca2+ interact with the dye, but ‘no potentiation of the absorbance changes is observed on adding 10 mM ATP (Boehringer Lot 1018349) to 10 FM
PREPARATIONS
OF NUCLEOTIDES
91
Fe3+, Cu2+, or Mn2+, or 100 pM Ca*+. The spectrum of the dye exhibits biphasic behavior with increasing concentrations of Cu*+ in the absence of ATP. The absorbance change increases between 10 and 50 FM Cu*+ but decreases slightly between 50 and 100 j&M cLl*+ with a slight shift of the isobestic point. At 1 mM Cu*+ the absorbance trough has shifted from 622 to 636 nm, and the maximum absorbance difference is reduced by half, compared with 50 pM Cu*+. Inhibition
of Hexokinase
by Aluminum
The actual substrate for most ATPutilizing enzymes is the magnesium-ATP complex. The AP+ ion is likely to form a very tight complex with ATP, which might compete with Mg-ATP in some cases. Yeast hexokinase is inhibited by aluminum, and the inhibition is antagonized by raising the magnesium concentration. Thus, in the presence of 0.2 mM ATP (Boehringer Lot 1018349), 0.02 mM AlCl, causes 45% inhibition with 0.7 mM MgCl, (concentration of free Mg*+ = 0.5 mM) and 35% inhibition with 1.2 mM MgC12 (free Mg2+ = 1 mM). In the absence of aluminum the rate is unaffected by increasing the MgCl, concentration from 0.7 to 1.2 mM. If the aluminum concentration is increased to 0.1 mM, the inhibition is 72 and 56% in the presence of 0.7 and 1.2 mM MgCl,, respectively. However, part of this inhibition may simply be due to the aluminum tying up a portion of the ATP in an aluminum-ATP complex and thus reducing the amount of Mg-ATP available for use as substrate. If 0.1 mM aluminum binds an equal amount of ATP, then the substrate concentration would be reduced from 0.2 to 0.1 mM ATP. The possible contribution of this factor to the inhibition by aluminum can be assessed by comparing the rate in the presence of 0.1 mM AlCl, and 0.2 mM ATP with the rate of a control containing only 0.1 mM ATP. This sets a lower limit to the degree of direct inhibition by aluminum, or the Al-ATP
92
TORNHEIM,
GILBERT,
complex, of 61 and 40% in the presence of 0.7 and 1.2 mM MgC&, respectively. The steady-state rate in the presence of aluminum is about the same whether the reaction is started by adding hexokinase, or glucose, or magnesium. However, when the reaction is started by adding hexokinase or glucose, the rate in the presence of aluminum is faster initially and slows down to a constant rate over a period of l-2 min; when the reaction is started by adding magnesium, the rate is slower initially, and then speeds up to a constant rate over a period of about 2 min. In the absence of aluminum, the rate with 0.1 mM ATP is 71% of the rate with 0.2 mM ATP, consistent with a K, for ATP of about 0.2 mM (3). When 0.4 mM EDTA is added to a reaction mixture containing 0.2 mM ATP, 0.1 mM AlC&, and 1.2 mM MgC12, the rate first decreases to a level consistent with the reduction in the free magnesium concentration and then rises slowly. The gradual increase in rate presumably represents removal of the inhibitory aluminum, since addition of EDTA in the absence of aluminum has little effect. However, when 100 times the usual amount of hexokinase is added to a reaction mixture containing 0.2 mM ATP, 0.1 mM AlC&, and 0.7 mM MgClz, all the ATP is utilized within about 6 min. With 10 times the normal amount of hexokinase, 90% of the ATP is utilized in 40 min; the reaction proceeds steadily with no indication of a biphasic process which would reflect a slower utilization of a substantial portion of the ATP, as might be expected if dissociation of the AlATP complex becomes rate limiting. When present at a level equal to one-tenth the ATP concentration, aluminum does not inhibit myokinase, creatine kinase, or phosphofructokinase (at noninhibitory levels of ATP) by more than lo%, which is the degree of inhibition that might occur if the Al-ATP complex does not bind to the enzyme. The ATP concentrations were chosen to be near the K, values. The
AND
LOWENSTEIN
inhibition of pyruvate kinase by 10 mM ATP is also not affected significantly by addition of 1 mM A13+ and A13+ does not substitute for Mg2+ in the pyruvate kinase reaction. DISCUSSION
Metal binding dyes are useful for measuring the free concentrations of magnesium and calcium ions in biochemical systems. However, the results presented here add a cautionary note that the levels of metal contaminants present in commercial nucleotide preparations and, perhaps, in other biochemicals can interfere considerably with such measurements. This is especially true for aluminum ions, since chelation of such ions by adenine nucleotides increases rather than decreases the effect of the metal on the dye Eriochrome Blue (Fig. 1). On the other hand, a low specificity makes a dye useful for detecting the presence of metal contaminants which might influence enzymatic reactions. Hexokinase can be substantially inhibited by aluminum ions; however, note that the concentrations of aluminum ions used here were larger than the level of contamination found in commercial preparations of ATP. The antagonism of the aluminum inhibition by magnesium is consistent with the inhibitory form being the Al-ATP complex, but a direct effect of aluminum on the enzyme is not ruled out. In studies of the inhibition of several kinases by chromium-ATP, hexokinase was the most strongly inhibited, with a Ki of 0.35 pM; by comparison the Ki with creatine kinase was 140 pM (4). As observed here with aluminum, the hexokinase reaction in the presence of chromium-ATP slowed down over several minutes to a lower rate, which suggested tighter binding of the analog as a function of time (5). Aluminum is generally considered to be nontoxic, except when such large quantities are ingested that a substantial amount of phosphate is complexed and energy metabolism
METAL
CONTAMINANTS
IN COMMERCIAL
becomes affected (6). The normal blood level of aluminum is about 18 PM, whereas the toxic level is about 90 mM (7). Aluminum is found in many tissues (8,9) and may be of physiological import (10). Plants contain 10 to 30 mg aluminum/kg fresh weight. The daily dietary intake of aluminum is about 45 mg for adults in the United States (6). It is possible that some biochemical processes could be affected by moderate doses of aluminum without being easily detectable, ATP that is heavily contaminated with aluminum causes a greater spectral change of the dye than can be accounted for by the aluminum contamination alone, even considering the potentiating effect of ATP (Fig. 1). This suggests the presence of additional metal contaminants which, although present in smaller amounts than aluminum, could be more potent in their biochemical effects. Since rather high concentrations of ATP or ADP are required to potentiate the spectral change caused by aluminum, the possibility must be considered that the potentiation is due to some other contaminant in the adenine nucleotides. In view of the magnesium contamination found in some commercial preparations of ATP and ADP (Table 1), experiments purporting to show a lack of magnesium requirement for an ATP-utilizing reaction should include a determination of the presence or absence of magnesium in the particular nucleotide preparations used.
PREPARATIONS
OF NUCLEOTIDES
93
Note added in proof. Womack and Colowick (1 I) have recently reported inhibition of hexokinase by aluminum present as a contaminant in some commercial preparations of ATP and ADP. The inhibition by aluminum is much stronger at a pH lower than that used in our studies.
REFERENCES 1. Scarpa, A. (1974) Biochemistry 13, 2789-2794. Scarpa, A. (1976) in Ion and Enzyme Electrodes in Biology and Medicine (Kessler, M., Clark, L. C., Jr., Lubbers, D. W., Silver, I. A., and Simon, W., eds.), pp. 252-260, University Park Press, Baltimore. 3. Fromm, H. J., and Zewe, V. (1962)5. Biol. Chem. 2.
237, 3027-3032.
4. Janson, C. A., and Cleland, W. W. (1974) .I. Biol. Chem. 249, 2572-2574. 5. Danenberg, K. D., and Cleland, W. W. (1975) Biochemistry 14, 28-39. 6. Venugopal, B., and Luckey, T. D. (1978) Metal Toxicity in Mammals, Vol. 2, pp. 105-113, Plenum, New York. 7. Williams, D. R. (1971) The Metals of Life, p. 56, Van Nostrand Reinhold, London. 8. Tipton, I. H. (1960) in Metal-Binding in Medicine (Seven, M. J., and Johnson, L. A., eds.), pp. 27-42, Lippincott, Philadelphia. 9. Butt, E. M., Nusbaum, R. E., Gilmore, T. C., and DiDio, S. L. (1960) in Metal-Binding in Medicine (Seven, M. J., and Johnson, L. A., eds.), pp. 43-49, Lippincott, Philadelphia. 10. Weinberg, E. D. (196O)in Metal-Binding in Medicine (Seven, M. J., and Johnson, L. A., eds.). pp. 324-334, Lippincott, Philadelphia. 11. Womack, F. C., and Colowick, S. P. (1979) Proc. Natl. Acad. Sci. USA 16. 5080-5084.