TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

47,477-482

(I 979)

Evidence for Specific Lead&aminolevulinate Complex Formation by Carbon-13 Nuclear Magnetic Resonance Spectroscopy’*’ C. STUART BAXTER, HOWARD E. WEY, AND ALAN D. CARDIN Departments of Environmental Health and Biological Chemistry, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267 Received April 25, 1978; accepted August 4, 1978 Evidence for Specific Lead-S-aminolevulinate Complex Formation by Carbon-13 Nuclear Magnetic Resonance Spectroscopy. BAXTER, C. S., WEY, H. E., AND CARDIN, A. D. (1979). Toxicol. Appl. Pharmacol. 47, 477-482. Lead is known to have toxic effects on heme synthesis, being an especially potent, and, relative to other metals, specific inhibitor of the enzyme &aminolevulinate dehydratase (ALAD). The mechanism of inhibition of ALAD by lead is uncertain, but binding of lead to ALAD sulfhydryl groups has been assumed. Mechanisms involving lead interaction with the enzyme substrate (ALA) rather than the enzyme are also reasonable, but have not as yet been considered; therefore the interaction of lead acetate with ALA was investigated by carbon-13 NMR spectroscopy, a technique shown to be very powerful in examining ligand-metal interactions. Addition of lead acetate solution to 0.25 M ALA at pH 6.5 resulted in a pronounced shift of certain of the carbon resonances of the latter, a maximal effect occurring at an ALA : Pb concentration ratio of 2. No similar effects were observed with acetates of calcium, zinc, cadmium, silver, or mercury. Studies on the variation in shift of resonances from invididual carbon atoms with lead acetate concentration suggested that lead interacted most strongly in the region of the carboxyl and z-carbon atoms of ALA. The variation of shift with pH showed that at an ALA : Pb ratio of I the pK, of the ALA carboxyl group was decreased by I .2 units. These studies demonstrate that lead is specific among the metals tested in forming a complex with 6-aminolevulinic acid. Such complex formation may be important in the mechanism of lead toxicity on the heme biosynthetic pathway.

Lead has represented a human health hazard since antiquity, and remains an environmental component of wide and major concern, continuing to be the topic of wide discussion and a large body of current literature (Goyer and Rhyne, 1972; Waldren and Stofen, 1974; Nat. Acad. Sci.-Nat. Res. Count., 1972; Hammond, 1977). Major ’ This investigation was supported by Research Grant Nos. ES 00159 and 00127 from the National Institutes of Health. 2 Presented at the 17th Annual Meeting of the Society of Toxicology, San Francisco, California, March 1978 under the sponsorship of P. B. Hammond.

sites of lead toxicity are the nervous and hematogenic systems,and a very real concern currently exists concerning the deleterious effects of lead, especially from gasoline combustion products and lead ore processing, on the above systems in humans (Hammond, 1977). The biochemical basis of lead toxicity remains as yet unclear (Vallee and Ulmer, 1972). As well as being capable of forming complexes with aminoacids and mono- and polynucleotides, lead is a potent and specific inhibitor of several enzymes. One of the latter is b-aminolevulinic acid dehydratase (ALAD),

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004-008X/79/030477-06$02.00/0 Copyright 0 1919 by Academic Press. Inc. rights of reproduction in any form reserved. Printed in Great Britain

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RAXT~,~EY,

a component enzyme in the biosynthesis of hemoproteins and cytochromes, which catalyzes the synthesis of the monopyrrole porphobilinogen from S-aminolevulinic acid (ALA). The correlation between blood lead concentration and activity of this enzyme has been utilized very widely and is a valuable index of human lead exposure (Hernberg et al., 1970). Different mechanisms of ALAD inhibition by lead apparently operate in lysed erythrocytes from lead-treated animals, and in lysates from untreated animals to which lead salts have been added in vitro (Vergnano et al.. 1968, 1969). Both mechanisms have been proposed to involve the interaction of lead ions with sulfhydryl groups essentialfor enzyme activity, but this postulate appears insufficient in the light of the observation that metals with generally higher affinity for these ligands than lead, such as mercury, silver, and cadmium, are nonethelessless potent inhibitors of ALAD. Speculations on the mechanism of the above inhibition have as a whole focused exclusively on possible lead-enzyme interactions, but none have addressed the possibility of complex formation between the metal and the reaction substrate. Evidence is presented herein that lead forms a complex with S-aminolevulinate, whereas several other metal ions do not show similar behavior. Evidence for Pb-ALA complexes has been obtained from studies utilizing pulsed Fourier Transform carbon-l 3 nuclear magnetic resonance spectroscopy, a technique which has been used widely to characterize metal interactions with biological ligands.

METHODS

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CARDIN

diameter tubes and contained IO”,, deuterium oxide’ to provide the internal lock signal. A concentric 3-mm tube containing a I O0 aqueous solution of dioxane was placed inside the sample tube to provide an external reference. The D,O was freed of metal ion contamination by repeated extraction with 0.001 7; diphenylthiocarbazonej solution in carbon tetrachloride. &Aminolevulinic acid hydrochloride5 was converted to the nitrate salt by anion exchange with silver nitrate solution, and freed from metal ions by passage through Chelex6 chelating ion-exchange resin. Solutions of metal salts’ (ultrapure grade) and ALA were made up in distilled, deionized, deoxygenated water. Conversion of ALA to the nitrate form avoided precipitation of insoluble precipitates of basic lead salts in solutions containing lead above pH 4, but in no way affected the NMR spectra.8 The pH of solutions was adjusted by addition of 401( sodium deuterioxideJ solution or glacial acetic acid. Chemical shifts were measured relative to the dioxane external standard and were reproducible to within +O.l ppm.

RESULTS The Fourier Transform natural abundance carbon-l 3 NM R spectrum of &aminolevulinic acid (Sigma, 0.25 M), measured under proton-decoupling conditions at pH 6.5 and 20 MHz, is shown in Fig. 1. Assignment of the resonances was made by comparison of chemical shifts with those of compounds of analogous structure, and verified by observations of relative pH dependencies. The variation with pH of the chemical shifts is shown in Fig. 2 for the resonances of the carbon-l 3 isotope at the a, /I, y, and carboxyl positions of ALA. In accordance with the chemical shift assignment of the 6 carbon resonance, the latter was found to be completely insenstitive to pH. ALA chemical shifts were also found to be independent of concentration between 0.1 and 1.0 M. In the presenceof an equimolar concentration of

Natural abundance carbon- I3 nuclear magnetic resonance spectra were obtained on a Varian AssoJ Merck and Co., Inc. Rahway, N.J. ciates CFT-20 instrument operating at a frequency of -1 Fisher Scientific, Fair Lawn, N.J. 20 MHz. An acquisition time of 0.51 I set and a pulse 5 Sigma Chemical Co., St. Louis, MO. delay of either 2.0 set for work at pH ~4 or 0.4 set 6 Bio-Rad Laboratories, Richmond, Calif. for work at pH values >4 was employed. Spectra ’ Alfa Products, Danvers, Mass. were collected in the Fourier Transform mode at a ’ Foilowing anion exchange the ALA concentration probe temperature of 30°C with proton decoupling. was redetermined using a spectrometric assay Sample solutions of I-ml volume were run in g-mm(Mauzerall and Granick, 1956).

LEAD-&AMINOLEVULINATE

Y

c2 Y-B=

-CO*H

H3NCH2COCH2CH2C02H

INTERACTIONS

NO;

I

I

I

200

150

I00

FIG. 1. Twenty-megahertz Fourier Transform proton-decoupled nuclear magnetic resonance spectrum of ALA at 3O”C, pH 6.5.

479

dioxone

I

50

I

0 Lvm

natural abundance carbon-l 3

divalent lead as the acetate complex salt, the pH curves were extensiveIy displaced to lower pK values (Fig. 2) relative to those for free ALA. The maximum effect was observed for the resonances corresponding to the carboxyl and a carbon atoms of ALA, although lessereffects were also noted for the j3 and y resonances. Quantitatively similar results were obtained using lead nitrate or perchlorate salts, instead of the acetate complex, up to pH 4. Above this pH the solutions deposited insoluble precipitates, presumably of basic lead salts, since solutions containing no ALA behaved in the sameway. Addition of sodium acetate at a concentration of 0.25 M neither resulted in displacement of the pH dependence curves of ALA, nor ALA in the presence of an equimolar concentration of lead acetate. These findings confirmed that the observed effects on pH -40 I I I I I , I profiles were not due to the acetate ion, and 0 1 2 3 4 5 6 7 pH demonstrate the stability of the ALA-lead complex towards the latter, which also forms FIG. 2. Variation of chemical shifts as a function of metal chelates. The ALA pH-dependence pH of the carbon-13 resonances of ALA (0.25 M) in curves were not displaced in the presence of the presence and absence of 0.25 M lead acetate.

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equimolar concentrations of calcium. zinc. cadmium, or silver acetates. Solutions containing ALA and mercuric acetate underwent a rapid reaction, with deposition of metallic mercury, within a period of 1 hr at 3O’C, the ambient probe temperature. This reaction was accompanied by the formation of apparently a single product, whose structure is presently being determined. The stoichiometry of the lead S-aminolevulinate complex was investigated by incremental additions of lead acetate to an aqueous solution of ALA nitrate maintained at a constant pH of 5.5. The carbon-13 resonances which were sensitive to lead showed increasing changes in position with increasing lead concentration, the carboxyl and CI carbon resonances moving to lower, and the /I resonances to higher, fields, and eventually reached constant maximum values which were not affected by further increases in lead concentration (Fig. 3). The variation of chemical shifts indicated the formation of a lead-&aminolevulinate complex with a I : 2 metal : ligand stoichiometry. Quantitatively similar results were obtained at pH values up to 7 units, but the instability of ALA we observed above pH 6 made accurate measurements impracticable. At 30°C ALA apparently decomposed to a single product, of as yet unknown composition.

-30

I 0

0.2

0.4 PblALA

0 6 MOLAR

0.8

1.0

12

RATIO

FIG. 3. Dependence of the chemica: shifts of the carbon-13 resonances of ALA on concentration of added lead acetate at pH 5.5.

A’..D

CARDIS

DISCUSSION Changes in the chemical shifts of resonances in the carbon-l 3 NMR spectra of AL.4 provide evidence that divalent lead salts interact with ALA to form complex species which may contain a maximum of two molecules of ALA per lead atom. Similar changes are not induced by the acetates of cadmium, calcium, silver, sodium, or zinc, or with the acetate ion alone, suggesting that lead may have considerable specificity for interaction with ALA. From the relative magnitudes of the effects on different carbon resonances it could be inferred that the principal site of lead interaction with ALA was at the carboxyl- and a-carbon atoms, with perhaps a less important site at the It-carbon atom. Effects on the resonance of the latter were especially marked at lower pH. The presence of this third site could explain the apparent observed stability of lead-ALA complexes to acetate ion. The reduction of the pK, of the carboxylic acid group of ALA on addition of an equimolar concentration of lead further suggested that this group was the major site of metal binding, and that the latter occurred with displacement of a hydrogen ion. The ability of lead to interact with ALA suggests that such interactions may be significant in lead effects on the enzyme ALAD. Inhibition of ALAD could be conceived as resulting from lead-ALA complex formation, rendering the substrate unavailable, or from the formation of abortive enzyme-leadsubstrate tertiary complexes. Erythrocyte ALAD activity measured at pH 6.8 is reduced to about 20:;; of its activity in human subjects with a blood lead concentration of 50 Lcg/lOcI dl or 2.5 pM, relative to subjects with normal concentration (I 5-25pg/ 100 dl for urban dwellers). At the former concentration lead is in considerable molar excess over ALA if the values of the concentration of the latter in the erythrocyte are similar to those reported in plasma (15 rig/ml or 0.1 pM) (P. B. Hammond, personal com-

LEAD-&AMINOLEVULINATE

munication). Thus a role for lead-ALA interactions in inhibition of ALAD in viro is plausible on the above considerations if just a few percent of erythrocyte-bound lead is present in the cytoplasm, since our studies show each atom of lead to be theoretically capable of binding two ALA molecules. Since other metals which inhibit the activity of ALAD to a lesser degree than lead have little or no observed interaction with ALA, the specific interaction of lead with ALA may provide a rationale for the specific inhibitory action of lead toward ALAD. Conversely, the greater affinity of silver and cadmium for sulfhydryl ligands (Vallee and Ullmer, 1972) implies that the mechanism for lead inhibition of ALAD does not solely involve reaction with this type of ligand on the enzyme. Zinc has been found to have either inhibitory or stimulatory effects on ALAD, and to be able to partially reverse inhibition of the enzyme by lead (Finelli et al., 1975: Thompson et al., 1977). The concentrations of zinc required for this latter effect, however, were several orders of magnitude higher than that of lead present. Zinc appears to be necessary for optimal ALAD activity in zYro (Finelli et al., 1975) suggesting that, in agreement with our findings, this metal interacts primarily with the enzyme, rather than its substrate. A mechanism for zinc reversal of lead inhibition of ALAD involving displacement of ALA from its lead complex by the optimally active zinc-containing enzyme would be consistent with this postulate, as well as with involvement of lead-ALA complex formation in ALAD inhibition. It is also pertinent to note that the pH activity profile of ALAD in hemolysatesappearsto be dependent on the lead : zinc concentration ratio (Border et al., 1976). An apparent difference in pH activity profile of ALAD in hemolysates from subjects exposed to lead in riL$o, and from “normal” subjectsfollowing addition of lead salts in ritro, has also been observed (Hernberg and Nikkanen, 1972). Although this finding was proposed to reflect differences in lead-ALAD interactions ill riro

lNTERACTIONS

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and irt vitro, it is clear that changes in zinc! lead concentrations in the lysates due to in ri~o lead exposure provides an alternative rationale. In support of this, exposure of rats to lead has been shown to reduce blood zinc concentrations (El-Gazzar et a/., 1978). Studies on the mechanisms of inhibition of ALAD by metal ions have so far mainly concentrated on tissue homogenates or hemolysates, both complex systems in which firm conclusions are difficult to reach. In addition to ALAD, its substrate ALA and toxic metals such as lead, there are other essential metal ions, including zinc and calcium, as well as numerous species which may alter mutual interactions between the metal and biochemical components. Hemolysates from lead-exposed humans may also well contain biochemical species lacking in normal hemolysates, or in different concentrations. Hence the reversal of ill c.ic:oleadinduced ALAD inhibition by heat may involve the release or formation of a species with high affinity for lead, effectively removing it from the ALAD environment. If such a speciesis absent, or in much lower concentration in blood from normal subjects, no relief of ALAD inhibition of lead added in vitro as has been observed experimentally (Hernberg and Nikkanen, 1972) would occur. Elucidation of precise mechanismsof leadALAD inhibition will clearly require additional detailed if) ritro experiments to determine the relative importance of interactions between the metal, pure enzyme, substrate, and other well-defined factors. Biophysical experiments of the above kind to determine further the significance of leadd-aminolevulinate complex formation in ALAD inhibition are in progress in this laboratory.

ACKNOWLEDGMENTS The authors gratefully acknowledge Drs. P. B. Hammond and V. N. Finelli for valuable discussions, and Mr. R. Froelich for technical assistance.

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REFERENCES BORDER,

E. A., CANTRELL, A. C., AND KILRO~T. A. (1976). The in vitro effect of zinc on the inhibition of human &aminolevulinic acid dehydratase by lead. Brir. J. /n&t. Med. 33, 85-87. EL-GAZZAR, R. M., FINELLI, V. N., BOIANO, J., AND PETERING, H. G. (1978). Influence of dietary zinc on lead toxicity in rats. To.&&. Lett. 1, 227-234. FINELLI, V. N., KLAUDER, D. S., KARAFFA, M. A., AND PETERING, H. G. (1975). Interaction of zinc and lead on S-aminolevulinate dehydratase. Biochem Biophys. Res. Cummrm. 65, 303-3 11. GOYER, R. A., AND RHYNE, B. C. (1972). Pathological effects of lead. Int. Rec. Palho/. 12, 2-78. HAMMOND, P. B. (1977). Exposure of humans to lead. Ann. Rev. Pharmacol. Toxicol. 17, 197-214. HERNBERG, S., AND NIKKANEN, J. (1972). Effect of lead on B-aminolevulinic acid dehydratase. A selective review. Pracor. L&X-. 24, 77-83. HERNBERG, S., NIKKANEN, J., MELLIN, G., AND LILLIUS, H. (1970). B-Aminolevulinic acid dehydrase as a measure of lead exposure. Arch. Enciron. Health 21, 140-145. MAUZERALL, D., AND GRANICK, S. (1956). The ocSMITH,

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currence and determination of d-aminolevulinic acid and porphobilinogen in urine. J. Biol. Chem. 219, 435-446. NAT. ACAD. SCI.~NAT. RES. COUNT. (1972). Airborne Lead in Perspective. Nat. Acad. Sci., Washington, D.C. THOMPSOX:. J., JONES, D. D., AND BEASLEY, W. H. (1977). The effect of metal ions on the activity of &aminolevuiinic acid dehydratase. Brit. J. Indttstst. Med. 34, 32-36. VALLEE, B. L., AND ULLMER, D. D. (1972). Biochemical effects of mercury, cadmium and lead. Ann. Rev. Biochem. 41, 91-127. VERGNANO, C., CASTEGNA, C., AND ARDOINO, V. (1969). Meccanismj di inibizione dell’attivito’ d‘-amino-levulinico deidritasica eritrocitaria nell’ intossicasione da piombo umana e sperimentale. Med. Lat. 60, 505-516. VERGNANO, C., CASTEGNA, C., AND BONSIGNORE, D. (1968). Regolazione allosterica della attivita S-amino-levulinico-deidratasica eritrocitaria. BUN. Sot. Ital. Biol. Sper. 64, 692-699. WALDRON, H. A., and STOFEN, D. (1974). Subclinical Lead Poisoning. Academic Press, New York and London.

Evidence for specific lead-delta-aminolevulinate complex formation by carbon-13 nuclear magnetic resonance spectroscopy.

TOXICOLOGY AND APPLIED PHARMACOLOGY 47,477-482 (I 979) Evidence for Specific Lead&aminolevulinate Complex Formation by Carbon-13 Nuclear Magneti...
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