BIOLOGICAL TRACE ELEMENT RESEARCH 4, 35-43 (1982)
Macromolecular Interactions with Cadmium and the Effects of Zinc, Copper, Lead, and Mercury Ions M. P. VERMA,t R. P. SHARMA,* AND D. R. BOURCIER Toxicology Program, Utah State University, Logan, UT 84322
Received March 6, 1981; Accepted September 10, 1981
Abstract Interactions of cadmium (Cd) ions with bovine serum albumin (BSA), bovine hepatic metallothionein (MT), calf thymus histone and deoxyribonucleic acid (DNA), and bovine hepatic chromatins were studied in the presence and absence of divalent zinc (Zn), copper (Cu), mercury (Hg), or lead (Pb) ions, using equilibrium dialysis at pH 7 and at 37~ The BSA had 3.5 Cd-binding sites with an apparent affinity constant of 1 x 105. The other metal ions inhibited the binding by reducing the affinity constant and the number of Cd-binding sites in BSA. There were 6 high affinity and 13 low affinity Cd-binding sites in the MT. Zinc ions had poor efficacy in reducing the binding of Cd to the MT. However, the Cu 2+ and Hg 2+ ions inhibited the Cd binding to a considerable extent, the former ions being more potent in this respect. Histone did not bind Cd. There were two kinds of Cd-binding sites in DNA: One mole of Cd per four moles DNA-phosphorus at low affinity sites, and one mole of Cd per 6.7 moles DNA-phosphorus at high affinity sites. Their apparent association constants were 8.3 • 105 and 4.4 x 106M, respectively. The other metal ions had inhibitory effects on the binding of Cd to DNA. Histone reduced the Cd-DNA interactions to only a minor extent. The other metal ions reduced the binding of Cd to DNA-histone complex to a small extent. Cadmium binds to the euchromatin (Euch), heterochromatin (Het), and Euch-Het mixture almost equally. The other metal ions reduced the binding maximally in Euch-Het followed next in order by Het and Euch. Cupric ions were the most potent inhibitors of the interactions of Cd with the nuclear materials.
Index Entries: Cadmium, metal-macromolecular interactions of; equilibrium dialysis; heavy metal interactions, in Cd-macromolecular binding; metal binding protein; tCurrent address: College of Veterinary Medicine, Haryana Agricultural University, Hissar, India. 9 1982 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163~,984/82/0300~)03552.00
35
36
VERMA, SHARMA, AND BOURCIER
zinc, interaction with Cd-protein complexes, cadmium displacement by metals; copper, interactions with Cd-protein compounds; mercury, interactions with Cd-protein compounds; macromolecular interactions with cadmium; lead, interactions with Cd-macromolecular compounds.
Introduction Cadmium (Cd) is a toxic metal that occurs in the animal body without any known biological function. The metal binds to various intracellular and extracellular macromolecules in the animal body. These interactions are considered to play important roles in the metabolism and toxicity of the metal (1-3). Other metal ions tend to compete with Cd for binding to the biomacromolecules and hence may alter its fate in the animal body. For example, administration of zinc (Zn), copper (Cu), and mercury (Hg) salts leads to an increase in hepatic Cd-uptake, but a decrease in renal uptake of the metal. Furthermore, Zn ions either abolish or diminish certain adverse biological effects of Cd, such as Cd-induced parakeratosis in calves, testicular necrosis and neoplasia, anemia, weight loss, hypertension, and suppression of mitochrondrial oxidative phosphorylation (4, 5). Interest in the toxicology of Cd has been growing in recent years. It is, therefore, important to know the details of its interactions with certain biomacromolecules and their complexes. Such information will be useful in further understanding the mechanism of its toxic action at the molecular level. Since the animals are simultaneously exposed to many different metals under natural conditions, it will be useful to know the effects of other metal ions on such interactions. In an earlier study (6) we reported that administration of cadmium in various domestic animals caused a greater rise in renal Cd and MT concentrations than those in liver. The displacement of Cd from liver by other dietary metals was indicated. The present investigations were undertaken to evaluate the interactions of Cd with bovine macromolecules, namely, serum albumin, metallothionein (MT), deoxyribonucleic acid (DNA), histone, and nuclear chromatins in the presence and absence of divalent Zn, Cu, Hg, and lead (Pb) ions under in vitro conditions.
Methods Materials Metallothionein (Cd-MT) of single-band electrophoretic purity was isolated from bovine liver (7). Multiple forms of MT are present in bovine liver and only the predominant one with a high molecular weight (15,200 daltons) was employed for interaction studies. Calf thymus DNA sodium containing 8% phosphorus, calf thymus histone, and bovine serum albumin (BSA) were purchased. Chromatins were isolated from bovine hepatic nuclei as described below. Deionized water and reagent grade cadmium chloride, zinc chloride, cupric chloride, mercuric chloride, and lead nitrate were employed in the study. Spectrapor membrane tubings with
HEAVY METAL--MACROMOLECULAR INTERACTIONS
37
different molecular weight cut-off points corresponding to 3500 for MT, 6000-8000 for BSA and histone, and 12,000-14,000 for all other compounds were used for dialysis as indicated later.
Isolation of Chromatins Fresh liver tissues from fetal calves and adult cows were homogenized in a Thomas tissue grinder with Teflon pestle (clearance 0.25 mm) using four volumes (W/V) of 0.05M Tris-HC1, pH 7.4, buffer containing 0.25M sucrose, 0.025M KC1, 0.005M MgC12, 0.0002M CaClz, 0.5% triton X-100, 1 percent dimethyl sulfoxide (DMSO), and 0.5 mM phenyl methyl sulfonyl fluoride (PMSF). Nuclei were isolated as described (8). All the operations were performed at 4~ The nuclei were suspended in 0.25M sucrose solution and sonicated for 10 s at 20,000 cps, 110 V, and 660 W. The suspension was centrifuged at 500g for 10 min. The supernatant was centrifuged at 3500g for 20 min to give a pellet of heterochromatin (Het). Centrifugation of the supernatant at 78,000g for 1 h gave a pellet of euchromatin-heterochromatin mixture (Euch-Het). A white precipitate of euchromatin (Euch) was obtained on addition of two volumes of cold ethanol to the 78,000g supernatant (2, 9). DNA contents of the three chromatin fractions were determined using diphenylamine reagent (10).
Equilibrium Dialysis The MT and BSA were dissolved in 10 mM Tris-HC1 buffer, pH 7 at 37~ The molecular weight of the MT was assumed to be 15,200 daltons, and that of the BSA as 65,500 daltons (11). The DNA and histone were dissolved in 2 mM sodium chloride and the pH adjusted to 7 with dilute hydrochloric acid or sodium hydroxide solution. The chromatin fractions were suspended in 1.54 mM NaC1-0.5 mM PMSF, pH 7 (8). The membrane tubings were soaked overnight in 0.01 N nitric acid, washed thoroughly with water, and equilibrated with the respective pH 7 medium for several hours (12). Preliminary trials indicated the optimum time intervals used to reach an equilibrium for each system. Samples of 2 mL of 1% BSA (30.5 x 10 -8 mol), 1 mL of 0.1% MT (6.58 x 10 -8 mol), 2 mL of 0.2% histone, 1 mL of 0.2% DNA plus 1 mL of 0.2% histone (13), 1 mL of 0.2% DNA plus 1 mL of the salt solution, or 2 mL each of Euch, Het, and Euch-Het (75 txg DNA in each) were separately transferred to the dialysis tubes of appropriate pore sizes and having a knot at the center. Air bubbles were removed and the contents were pushed towards the center with twisting of the tubes. The open end was tied with a polyester thread. The two empty parts of the tubes on either sides of the samples were tied together with the thread. The dialysis bags loaded with the samples were suspended in test tubes containing 4 or 5 mL of appropriate medium. The threads were held in position with the help of stoppers to allow hanging of the samples inside the solvents. The solvents contained known amounts of Cd with or without divalent Zn, Cu, Hg, or Pb ions as indicated in the Results section. The tubes were incubated in a metabolic shaking water bath for 8, 11, and 16 h using 12,000-14,000, 6000-8000, and 3500 mw cut-offpoint membranes, respec-
38
VERMA, SHARMA, AND BOURCIER
tively. At the end of incubation, Cd was determined in the inner and outer compartments using atomic absorption spectrophotometry. Suspensions and viscous solutions were dissolved by the addition of concentrated nitric acid before analyzing for Cd. The concentration of the metal in the outer chamber represented free Cd, whereas the bound Cd was determined by the difference between the two sides. The dialysis bags did not have any measurable binding for Cd and hence no corrections were necessary. The data were analyzed as described by Scatchard (14) and the apparent linear parts of the relationships were fitted with a linear regression line. No corrections were made for the overlapping slopes, if more than one slope were apparent. In the case of nuclear preparations, the results are depicted as bar diagrams indicating mean and standard deviations.
Results A wide range of concentrations were used to study the interactions of cadmium with different macromolecules. For example, the initial concentrations of Cd in BSA interactions ranged from 10 -8 to 10-3M. At the lower concentrations of Cd, below 10-aM, the Scatchard plot did not provide a predictable relationship. During this range the moles of Cd bound per mole of BSA increased as the concentration of Cd was increased. Although there was a wide variability noticed in data, the trends were similar in several experiments. At the higher Cd concentrations the
~2
L
t
2
2
0~
' !
' 2 r
~
"~
3
"~ 0
9
i
2
3
r
Fig. 1. The Scatchard plot showing binding of C d 2+ to bovine serum albumin in the absence and presence of divalent metal ions: r = moles of ligand bound per mole of macromolecule, x = concentration of free Cd (M). The solid lines represent the regression curves relating to the points. The concentration of Cd (unbound) in the range represented above varied from 1.43 x 10 -4 to 2.86 • 10-4M. All other metals were employed in a fixed concentration of 1 • 10-4M. Cadmium alone (Q) has been shown on both figures for reference to its displacement by zinc (O), copper (A), lead (V]), and mercury (&).
39
HEAVY METAL--MACROMOLECULAR INTERACTIONS
Scatchard plot of Cd-BSA interactions provided an apparently linear relationship. Figure 1 illustrates the results of a representative experiment. When the Cd concentration was higher than 2.86 • 10-4M a saturation of all binding sites on this macromolecule resulted, indicating no change in the ~/value (moles of Cd bound per mole of BSA). The concentration of other divalent ions selected for BSA interaction study was closer to the lowest Cd concentration on the Scatchard plot. Since the evaluation of interactions was made by studying the displacement of Cd from binding sites, a large displacement at the low Cd concentrations was not desirable. In addition, because the divalent metals are known to cause precipitation of proteins and thus alter their structures, care was taken to select the metal concentrations. The other divalent ions were hence used at a fixed concentration of 10-4M. The metal concentrations used for other metals were similarly determined for other macromolecules. Figure 1 shows a Scatchard plot of BSA interactions with metals. The lines drawn through various points are the linear regression curves. Some of the points may have higher weights because more than one value is represented by them. For Cd-BSA interactions the intercept on the x-axis suggests 3.5 binding sites on each BSA molecule. The intercept on the y-axis indicates n/K, where n is the number of binding sites (intercept on the x axis) and K -- affinity constant (14) yielding an apparent affinity constant of 105M. Zinc, Hg 2+ and Pb 2+ ions inhibited the binding by lowering the association constant with efficacy decreasing in that order. The Cu 2+ ions acted by reducing the number of binding sites. 8I
x
x
4
2
4
6
8
10
12
r
Fig. 2. The Scatchard plot showing interactions of C d 2+ with bovine hepatic metallothionein in the absence and presence of other divalent cations: ~/ = ratio of Cd and MT bound at a given concentration of Cd, x = concentration of free Cd. Cd alone, 9.6 x 10-5 to 1.92 • 10-4M (O); Cd in the range of 3.2 x 10-5 to 8.0 • 10-SM in the presence of 1.6 x 10 - 4 zinc (9 3.84 • 10-SM copper (A), and 3.84 x 10-SM mercury (A). Lead in several concentrations did not show a specific displacement of Cd from MT.
40
VERMA, SHARMA, AND BOURCIER
9
-x
0
t
0.1
0.2
4 k,,.
0.1
0.2 r
Fig. 3.
The Scatchard plot showing Cd 2§ binding per unit phosphorus of calf thymus
DNA in the absence and presence of divalent cations (4.29 x 10-4M). Cadmium concentration ranged from 1.43 x 10-4 to 5.72 x 10-4M and shown alone ( I ) , in the presence of zinc (O), copper (zS), lead ([~) and mercury (A); r = moles of Cd bound per mole of phosphorus, x = molar concentration of free Cd in the system. The Scatchard plot (Fig. 2) of C d - M T interactions had two slopes. The intercept of the steeper portion of the plot on the x-axis indicated six high affinity sites, whereas 13 low affinity sites were indicated by the intercept of the other portion of the plot. In the presence of high concentration of Zn 2+ ions, the high affinity line disappeared, but the low affinity slope did not change. This indicated that the Zn 2+ ions displaced Cd 2+ from the high affinity sites without affecting the low affinity sites. The Cu z+ and Hg 2+ inhibited Cd-binding to MT at low affinity sites, and lowered the association constant at high affinity sites, the Cu 2§ ions were more potent in this respect. The Scatchard plot (Fig. 3) of Cd-DNA interactions also had two slopes. Their intercepts on the x-axis indicated binding of I mol Cd/4 mol phosphorus (P) at low affinity sites, and 1 mol Cd/6.7 mol P at high affinity sites. The association constants, as calculated from the intercepts on the y-axis, were 8.3 x 105M and 4.4 x 106M, respectively. The other metal ions inhibited the binding of Cd at higher
HEAVY
METAL--MACROMOLECULAR
41
INTERACTIONS
IO0
I.O
F v
o m
DNA
DNA-HI STONE
Fig. 4. Binding of Cd 2+ to DNA and to DNA-histone complex in the absence and presence of divalent Zn, Cu, Pb, or Hg ions (1.67 x 10-aM). Histone did not bind Cd. Intervals on bars represent standard deviations of 3-6 observations. affinity sites, and reduced the association constant of low affinity sites with efficacy: Cu 2+ = Pb 2+ > Hg 2+ > Zn 2+ The bar diagrams of C d - D N A and Cd-DNA-histone interactions (Fig. 4) indicate that the DNA binds Cd, but the histone does not. In a 1 : 1 DNA-histone complex the binding of Cd to DNA was reduced by only 2%. Divalent Zn, Cu, Hg, and Pb ions decreased Cd binding to DNA and DNA-histone complex to a small extent. However, they had potent effects in the chromatins as shown in Fig. 5. The Cu 2+ ions were most potent in this respect. Inhibitory effect of the metals on Cd-chromatin interactions decreased in the order: Euch-Het > Het > Euch. IOO
x
(zk. I t~
'-' 50 ==
H iN EUCHROMATIN
EUCHROMATINHETEROCHROMATIN HETEROCHROMATIN
Fig. 5. Effect of equimolar concentations (1.33 x 10-SM) of divalent Zn, Cu, Pb, or Hg ions on binding of Cd 2§ to bovine hepatic euchromatin, heterchromatin, and their mixture (75 Ixg DNA in each). Intervals on bars represent standard deviations of 3-6 observations,
42
VERMA, SHARMA, AND BOURCIER
Discussion Detection of 3.5 Cd-binding sites in BSA in this study is close to the value of 3 reported by Rao and Lal (15). The metal ions (Zn, Cu, Hg, and Pb) are effective in displacing Cd from the BSA. Rao and Lal have likewise reported displacement of the metal by Zn and Cu ions. It is possible that the Scatchard plot of Cd-BSA interaction had more than one slope. The deviation from linearity was apparent at the low concentrations of Cd where a cooperative effect in Cd binding to BSA was observed. The bovine hepatic MT (15,200 daltons) contained 13 Cd-binding sites as against the reported values of 6 to 7 sites in other MT (16, 17). The difference seems to arise from the fact that the bovine hepatic MT molecules consisted of two monomers joined together (7). The higher molecualr weight hepatic MT was used since it was the predominant metal binding protein (7). The presence of high and low affinity sites in the MT is surprising because the cysteine residues are reported to be the only Cd-binding sites in MT (18). The second kind of sites in the bovine hepatic MT remain unknown at present, but can be accounted for by the presence of larger amount of glutamate in bovine MT than in equine renal MT (7, 19). The Cd-binding constants for the two sites were not calculated because the interactions may occur in the presence of Zn and Cu ions present in the MT. Only high concentrations of Zn ions are able to compete with Cd for binding to the MT as reported by Kagi and Vallee (16). Cadmium is not able to interact with the low affinity sites of MT in the presence of Cu 2+ and Hg 2§ ions. The cupric ions were more effective than mercuric ions in displacing Cd from the high affinity sites, which is surprising. The binding of 1 mole of Cd to 4 moles of phosphate in the DNA is identical to the binding of 1 Mg atom to 4 moles of phosphate (20). The presence of two kinds of Cd-binding sites in DNA supports the finding of others that the phosphate and base-nitrogens are involved in metal-DNA interactions (21-24). Considering that there is only a 2% reduction in the interactions of Cd with the DNA-histone complex, it may safely be presumed that the histone does not play any significant role in Cd-DNA interactions. Cadmium binds to the Euch and Het to almost the same extent. In vivo studies have, however, indicated that the metal ions bind primarily to the latter (25). Divalent Zn, Cu, Hg, and Pb ions prevent the interactions of Cd with the nuclear materials, Cu ions being most potent in this respect.
Acknowledgments The research reported here was supported in part by USPHS contract FDA 223-74-4195. Approved and published as Utah State University Agricultural Experiment Station Journal Paper No. 2450.
HEAVY METAL--MACROMOLECULAR
INTERACTIONS
43
References 1. J. F. Moore, R. A. Goyer, and M. Wilson, Lab. Invest. 29, 488 (1973). 2. S. E. Bryan, C. Lambert, K. J. Hardy, and S. Simons, Science 18 6 832 (1974). 3. M. Friedman, Protein Metal Interaction, Plenum Press, New York, 1974, pp. 289-328. 4. L. Friberg, M. Piscator, C. F. Nordberg, and T. Kjellstrom, Cadmium in the Environment, 2nd ed., CRC Press, Cleveland, Ohio, 1974. 5. H. H. Sanstead, in Effects and Dose-Response Relationships of Toxic Metals, G. F. Nordberg, ed., Elsevier, New York, 1976, pp. 511-526. 6. M. P. Verma, R. P. Sharma, and J. C. Street, Am. J. Vet. Res. 3 9, 1911 (1978). 7. R. P. Sharma and M. P. Verma, Am. J. Vet. Res. 41, 548 (1980). 8. K.J. Hardy and S. E. Bryan, in Biological Implications of Metals in the Environment, H. Drucher and R. E. Wildung, eds., US ERDA, Virginia , 1977, pp. 488-506. 9. W. G. Yasmineh and J. J. Yunis, Biochem. Biophys. Res. Commun. 35, 779 (1969). 10. W. D. Armstrong and C. W. Cart, Physical Chemistry, Laboratory Directions, Burgess Publishing, Minneapolis, Minnesota, 1963, pp. 112-113. 11. T. P. King and M. Spencer, J. Biol. Chem. 245, 6134 (1970). 12. T. R. Hughes, and I. M. Klotz, in Methods of Biochemical Analysis, D. Glick, ed., Interscience, New York, 1956, pp. 265-299. 13. K. Y. Chang and C. W. Carr, Biochem. Biophys. Acta. 157, 127 (1968). 14. G. Scatchard, Ann. N. Y. Acad. Sci. 51, 660 (1949). 15. M.S.M. Rao and H. Lal, J. Amer. Chem. Soc. 80, 3226 (1958). 16. J. H. R. Kagi and B. L. Vallee, J. Biol. Chem. 236, 2435 (1961). 17. P. Pulido, J. H. R. Kagi, and B. L. Vallee, Biochem. 5, 1768, (1966). 18. J. H. R. Kagi and B. L. Vallee, J. Biol. Chem. 235, 3460 (1960). 19. Y. Kojima, C. Berger, B. L. Vallee, and J. H. R. Kagi, Proc. Natl. Acad. Sci. USA 73, 3413 (1976). 20. H. R. Mahler and E. H. Cordes, Biological Chemistry, 2nd ed., Harper and Row, New York, 1971, p. 189. 21. T. Yamane and N. Davidson, J. Amer. Chem. Soc. 83, 2599 (1961). 22. G. L. Eichhorn, Advan. Chem. Ser. 62, 378 (1961). 23. Y. A. Shin and G. L. Eichhom, Biochemistry 7, 1026 (1968). 24. H. Richard, J. P. Schrieber, and M. Daune, Biopolymers 12, 1 (1973). 25. V. Mizuhira and M. Kimura, Proc. Elec. Microsc. Soc. Amer. 31,402 (1973).
Macromolecular Interactions with Cadmium and the Effects of Zinc, Copper, Lead, and Mercury Ions M. P. VERMA,t R. P. SHARMA,* AND D. R. BOURCIER Toxicology Program, Utah State University, Logan, UT 84322
Received March 6, 1981; Accepted September 10, 1981
Abstract Interactions of cadmium (Cd) ions with bovine serum albumin (BSA), bovine hepatic metallothionein (MT), calf thymus histone and deoxyribonucleic acid (DNA), and bovine hepatic chromatins were studied in the presence and absence of divalent zinc (Zn), copper (Cu), mercury (Hg), or lead (Pb) ions, using equilibrium dialysis at pH 7 and at 37~ The BSA had 3.5 Cd-binding sites with an apparent affinity constant of 1 x 105. The other metal ions inhibited the binding by reducing the affinity constant and the number of Cd-binding sites in BSA. There were 6 high affinity and 13 low affinity Cd-binding sites in the MT. Zinc ions had poor efficacy in reducing the binding of Cd to the MT. However, the Cu 2+ and Hg 2+ ions inhibited the Cd binding to a considerable extent, the former ions being more potent in this respect. Histone did not bind Cd. There were two kinds of Cd-binding sites in DNA: One mole of Cd per four moles DNA-phosphorus at low affinity sites, and one mole of Cd per 6.7 moles DNA-phosphorus at high affinity sites. Their apparent association constants were 8.3 • 105 and 4.4 x 106M, respectively. The other metal ions had inhibitory effects on the binding of Cd to DNA. Histone reduced the Cd-DNA interactions to only a minor extent. The other metal ions reduced the binding of Cd to DNA-histone complex to a small extent. Cadmium binds to the euchromatin (Euch), heterochromatin (Het), and Euch-Het mixture almost equally. The other metal ions reduced the binding maximally in Euch-Het followed next in order by Het and Euch. Cupric ions were the most potent inhibitors of the interactions of Cd with the nuclear materials.
Index Entries: Cadmium, metal-macromolecular interactions of; equilibrium dialysis; heavy metal interactions, in Cd-macromolecular binding; metal binding protein; tCurrent address: College of Veterinary Medicine, Haryana Agricultural University, Hissar, India. 9 1982 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163~,984/82/0300~)03552.00
35
36
VERMA, SHARMA, AND BOURCIER
zinc, interaction with Cd-protein complexes, cadmium displacement by metals; copper, interactions with Cd-protein compounds; mercury, interactions with Cd-protein compounds; macromolecular interactions with cadmium; lead, interactions with Cd-macromolecular compounds.
Introduction Cadmium (Cd) is a toxic metal that occurs in the animal body without any known biological function. The metal binds to various intracellular and extracellular macromolecules in the animal body. These interactions are considered to play important roles in the metabolism and toxicity of the metal (1-3). Other metal ions tend to compete with Cd for binding to the biomacromolecules and hence may alter its fate in the animal body. For example, administration of zinc (Zn), copper (Cu), and mercury (Hg) salts leads to an increase in hepatic Cd-uptake, but a decrease in renal uptake of the metal. Furthermore, Zn ions either abolish or diminish certain adverse biological effects of Cd, such as Cd-induced parakeratosis in calves, testicular necrosis and neoplasia, anemia, weight loss, hypertension, and suppression of mitochrondrial oxidative phosphorylation (4, 5). Interest in the toxicology of Cd has been growing in recent years. It is, therefore, important to know the details of its interactions with certain biomacromolecules and their complexes. Such information will be useful in further understanding the mechanism of its toxic action at the molecular level. Since the animals are simultaneously exposed to many different metals under natural conditions, it will be useful to know the effects of other metal ions on such interactions. In an earlier study (6) we reported that administration of cadmium in various domestic animals caused a greater rise in renal Cd and MT concentrations than those in liver. The displacement of Cd from liver by other dietary metals was indicated. The present investigations were undertaken to evaluate the interactions of Cd with bovine macromolecules, namely, serum albumin, metallothionein (MT), deoxyribonucleic acid (DNA), histone, and nuclear chromatins in the presence and absence of divalent Zn, Cu, Hg, and lead (Pb) ions under in vitro conditions.
Methods Materials Metallothionein (Cd-MT) of single-band electrophoretic purity was isolated from bovine liver (7). Multiple forms of MT are present in bovine liver and only the predominant one with a high molecular weight (15,200 daltons) was employed for interaction studies. Calf thymus DNA sodium containing 8% phosphorus, calf thymus histone, and bovine serum albumin (BSA) were purchased. Chromatins were isolated from bovine hepatic nuclei as described below. Deionized water and reagent grade cadmium chloride, zinc chloride, cupric chloride, mercuric chloride, and lead nitrate were employed in the study. Spectrapor membrane tubings with
HEAVY METAL--MACROMOLECULAR INTERACTIONS
37
different molecular weight cut-off points corresponding to 3500 for MT, 6000-8000 for BSA and histone, and 12,000-14,000 for all other compounds were used for dialysis as indicated later.
Isolation of Chromatins Fresh liver tissues from fetal calves and adult cows were homogenized in a Thomas tissue grinder with Teflon pestle (clearance 0.25 mm) using four volumes (W/V) of 0.05M Tris-HC1, pH 7.4, buffer containing 0.25M sucrose, 0.025M KC1, 0.005M MgC12, 0.0002M CaClz, 0.5% triton X-100, 1 percent dimethyl sulfoxide (DMSO), and 0.5 mM phenyl methyl sulfonyl fluoride (PMSF). Nuclei were isolated as described (8). All the operations were performed at 4~ The nuclei were suspended in 0.25M sucrose solution and sonicated for 10 s at 20,000 cps, 110 V, and 660 W. The suspension was centrifuged at 500g for 10 min. The supernatant was centrifuged at 3500g for 20 min to give a pellet of heterochromatin (Het). Centrifugation of the supernatant at 78,000g for 1 h gave a pellet of euchromatin-heterochromatin mixture (Euch-Het). A white precipitate of euchromatin (Euch) was obtained on addition of two volumes of cold ethanol to the 78,000g supernatant (2, 9). DNA contents of the three chromatin fractions were determined using diphenylamine reagent (10).
Equilibrium Dialysis The MT and BSA were dissolved in 10 mM Tris-HC1 buffer, pH 7 at 37~ The molecular weight of the MT was assumed to be 15,200 daltons, and that of the BSA as 65,500 daltons (11). The DNA and histone were dissolved in 2 mM sodium chloride and the pH adjusted to 7 with dilute hydrochloric acid or sodium hydroxide solution. The chromatin fractions were suspended in 1.54 mM NaC1-0.5 mM PMSF, pH 7 (8). The membrane tubings were soaked overnight in 0.01 N nitric acid, washed thoroughly with water, and equilibrated with the respective pH 7 medium for several hours (12). Preliminary trials indicated the optimum time intervals used to reach an equilibrium for each system. Samples of 2 mL of 1% BSA (30.5 x 10 -8 mol), 1 mL of 0.1% MT (6.58 x 10 -8 mol), 2 mL of 0.2% histone, 1 mL of 0.2% DNA plus 1 mL of 0.2% histone (13), 1 mL of 0.2% DNA plus 1 mL of the salt solution, or 2 mL each of Euch, Het, and Euch-Het (75 txg DNA in each) were separately transferred to the dialysis tubes of appropriate pore sizes and having a knot at the center. Air bubbles were removed and the contents were pushed towards the center with twisting of the tubes. The open end was tied with a polyester thread. The two empty parts of the tubes on either sides of the samples were tied together with the thread. The dialysis bags loaded with the samples were suspended in test tubes containing 4 or 5 mL of appropriate medium. The threads were held in position with the help of stoppers to allow hanging of the samples inside the solvents. The solvents contained known amounts of Cd with or without divalent Zn, Cu, Hg, or Pb ions as indicated in the Results section. The tubes were incubated in a metabolic shaking water bath for 8, 11, and 16 h using 12,000-14,000, 6000-8000, and 3500 mw cut-offpoint membranes, respec-
38
VERMA, SHARMA, AND BOURCIER
tively. At the end of incubation, Cd was determined in the inner and outer compartments using atomic absorption spectrophotometry. Suspensions and viscous solutions were dissolved by the addition of concentrated nitric acid before analyzing for Cd. The concentration of the metal in the outer chamber represented free Cd, whereas the bound Cd was determined by the difference between the two sides. The dialysis bags did not have any measurable binding for Cd and hence no corrections were necessary. The data were analyzed as described by Scatchard (14) and the apparent linear parts of the relationships were fitted with a linear regression line. No corrections were made for the overlapping slopes, if more than one slope were apparent. In the case of nuclear preparations, the results are depicted as bar diagrams indicating mean and standard deviations.
Results A wide range of concentrations were used to study the interactions of cadmium with different macromolecules. For example, the initial concentrations of Cd in BSA interactions ranged from 10 -8 to 10-3M. At the lower concentrations of Cd, below 10-aM, the Scatchard plot did not provide a predictable relationship. During this range the moles of Cd bound per mole of BSA increased as the concentration of Cd was increased. Although there was a wide variability noticed in data, the trends were similar in several experiments. At the higher Cd concentrations the
~2
L
t
2
2
0~
' !
' 2 r
~
"~
3
"~ 0
9
i
2
3
r
Fig. 1. The Scatchard plot showing binding of C d 2+ to bovine serum albumin in the absence and presence of divalent metal ions: r = moles of ligand bound per mole of macromolecule, x = concentration of free Cd (M). The solid lines represent the regression curves relating to the points. The concentration of Cd (unbound) in the range represented above varied from 1.43 x 10 -4 to 2.86 • 10-4M. All other metals were employed in a fixed concentration of 1 • 10-4M. Cadmium alone (Q) has been shown on both figures for reference to its displacement by zinc (O), copper (A), lead (V]), and mercury (&).
39
HEAVY METAL--MACROMOLECULAR INTERACTIONS
Scatchard plot of Cd-BSA interactions provided an apparently linear relationship. Figure 1 illustrates the results of a representative experiment. When the Cd concentration was higher than 2.86 • 10-4M a saturation of all binding sites on this macromolecule resulted, indicating no change in the ~/value (moles of Cd bound per mole of BSA). The concentration of other divalent ions selected for BSA interaction study was closer to the lowest Cd concentration on the Scatchard plot. Since the evaluation of interactions was made by studying the displacement of Cd from binding sites, a large displacement at the low Cd concentrations was not desirable. In addition, because the divalent metals are known to cause precipitation of proteins and thus alter their structures, care was taken to select the metal concentrations. The other divalent ions were hence used at a fixed concentration of 10-4M. The metal concentrations used for other metals were similarly determined for other macromolecules. Figure 1 shows a Scatchard plot of BSA interactions with metals. The lines drawn through various points are the linear regression curves. Some of the points may have higher weights because more than one value is represented by them. For Cd-BSA interactions the intercept on the x-axis suggests 3.5 binding sites on each BSA molecule. The intercept on the y-axis indicates n/K, where n is the number of binding sites (intercept on the x axis) and K -- affinity constant (14) yielding an apparent affinity constant of 105M. Zinc, Hg 2+ and Pb 2+ ions inhibited the binding by lowering the association constant with efficacy decreasing in that order. The Cu 2+ ions acted by reducing the number of binding sites. 8I
x
x
4
2
4
6
8
10
12
r
Fig. 2. The Scatchard plot showing interactions of C d 2+ with bovine hepatic metallothionein in the absence and presence of other divalent cations: ~/ = ratio of Cd and MT bound at a given concentration of Cd, x = concentration of free Cd. Cd alone, 9.6 x 10-5 to 1.92 • 10-4M (O); Cd in the range of 3.2 x 10-5 to 8.0 • 10-SM in the presence of 1.6 x 10 - 4 zinc (9 3.84 • 10-SM copper (A), and 3.84 x 10-SM mercury (A). Lead in several concentrations did not show a specific displacement of Cd from MT.
40
VERMA, SHARMA, AND BOURCIER
9
-x
0
t
0.1
0.2
4 k,,.
0.1
0.2 r
Fig. 3.
The Scatchard plot showing Cd 2§ binding per unit phosphorus of calf thymus
DNA in the absence and presence of divalent cations (4.29 x 10-4M). Cadmium concentration ranged from 1.43 x 10-4 to 5.72 x 10-4M and shown alone ( I ) , in the presence of zinc (O), copper (zS), lead ([~) and mercury (A); r = moles of Cd bound per mole of phosphorus, x = molar concentration of free Cd in the system. The Scatchard plot (Fig. 2) of C d - M T interactions had two slopes. The intercept of the steeper portion of the plot on the x-axis indicated six high affinity sites, whereas 13 low affinity sites were indicated by the intercept of the other portion of the plot. In the presence of high concentration of Zn 2+ ions, the high affinity line disappeared, but the low affinity slope did not change. This indicated that the Zn 2+ ions displaced Cd 2+ from the high affinity sites without affecting the low affinity sites. The Cu z+ and Hg 2+ inhibited Cd-binding to MT at low affinity sites, and lowered the association constant at high affinity sites, the Cu 2§ ions were more potent in this respect. The Scatchard plot (Fig. 3) of Cd-DNA interactions also had two slopes. Their intercepts on the x-axis indicated binding of I mol Cd/4 mol phosphorus (P) at low affinity sites, and 1 mol Cd/6.7 mol P at high affinity sites. The association constants, as calculated from the intercepts on the y-axis, were 8.3 x 105M and 4.4 x 106M, respectively. The other metal ions inhibited the binding of Cd at higher
HEAVY
METAL--MACROMOLECULAR
41
INTERACTIONS
IO0
I.O
F v
o m
DNA
DNA-HI STONE
Fig. 4. Binding of Cd 2+ to DNA and to DNA-histone complex in the absence and presence of divalent Zn, Cu, Pb, or Hg ions (1.67 x 10-aM). Histone did not bind Cd. Intervals on bars represent standard deviations of 3-6 observations. affinity sites, and reduced the association constant of low affinity sites with efficacy: Cu 2+ = Pb 2+ > Hg 2+ > Zn 2+ The bar diagrams of C d - D N A and Cd-DNA-histone interactions (Fig. 4) indicate that the DNA binds Cd, but the histone does not. In a 1 : 1 DNA-histone complex the binding of Cd to DNA was reduced by only 2%. Divalent Zn, Cu, Hg, and Pb ions decreased Cd binding to DNA and DNA-histone complex to a small extent. However, they had potent effects in the chromatins as shown in Fig. 5. The Cu 2+ ions were most potent in this respect. Inhibitory effect of the metals on Cd-chromatin interactions decreased in the order: Euch-Het > Het > Euch. IOO
x
(zk. I t~
'-' 50 ==
H iN EUCHROMATIN
EUCHROMATINHETEROCHROMATIN HETEROCHROMATIN
Fig. 5. Effect of equimolar concentations (1.33 x 10-SM) of divalent Zn, Cu, Pb, or Hg ions on binding of Cd 2§ to bovine hepatic euchromatin, heterchromatin, and their mixture (75 Ixg DNA in each). Intervals on bars represent standard deviations of 3-6 observations,
42
VERMA, SHARMA, AND BOURCIER
Discussion Detection of 3.5 Cd-binding sites in BSA in this study is close to the value of 3 reported by Rao and Lal (15). The metal ions (Zn, Cu, Hg, and Pb) are effective in displacing Cd from the BSA. Rao and Lal have likewise reported displacement of the metal by Zn and Cu ions. It is possible that the Scatchard plot of Cd-BSA interaction had more than one slope. The deviation from linearity was apparent at the low concentrations of Cd where a cooperative effect in Cd binding to BSA was observed. The bovine hepatic MT (15,200 daltons) contained 13 Cd-binding sites as against the reported values of 6 to 7 sites in other MT (16, 17). The difference seems to arise from the fact that the bovine hepatic MT molecules consisted of two monomers joined together (7). The higher molecualr weight hepatic MT was used since it was the predominant metal binding protein (7). The presence of high and low affinity sites in the MT is surprising because the cysteine residues are reported to be the only Cd-binding sites in MT (18). The second kind of sites in the bovine hepatic MT remain unknown at present, but can be accounted for by the presence of larger amount of glutamate in bovine MT than in equine renal MT (7, 19). The Cd-binding constants for the two sites were not calculated because the interactions may occur in the presence of Zn and Cu ions present in the MT. Only high concentrations of Zn ions are able to compete with Cd for binding to the MT as reported by Kagi and Vallee (16). Cadmium is not able to interact with the low affinity sites of MT in the presence of Cu 2+ and Hg 2§ ions. The cupric ions were more effective than mercuric ions in displacing Cd from the high affinity sites, which is surprising. The binding of 1 mole of Cd to 4 moles of phosphate in the DNA is identical to the binding of 1 Mg atom to 4 moles of phosphate (20). The presence of two kinds of Cd-binding sites in DNA supports the finding of others that the phosphate and base-nitrogens are involved in metal-DNA interactions (21-24). Considering that there is only a 2% reduction in the interactions of Cd with the DNA-histone complex, it may safely be presumed that the histone does not play any significant role in Cd-DNA interactions. Cadmium binds to the Euch and Het to almost the same extent. In vivo studies have, however, indicated that the metal ions bind primarily to the latter (25). Divalent Zn, Cu, Hg, and Pb ions prevent the interactions of Cd with the nuclear materials, Cu ions being most potent in this respect.
Acknowledgments The research reported here was supported in part by USPHS contract FDA 223-74-4195. Approved and published as Utah State University Agricultural Experiment Station Journal Paper No. 2450.
HEAVY METAL--MACROMOLECULAR
INTERACTIONS
43
References 1. J. F. Moore, R. A. Goyer, and M. Wilson, Lab. Invest. 29, 488 (1973). 2. S. E. Bryan, C. Lambert, K. J. Hardy, and S. Simons, Science 18 6 832 (1974). 3. M. Friedman, Protein Metal Interaction, Plenum Press, New York, 1974, pp. 289-328. 4. L. Friberg, M. Piscator, C. F. Nordberg, and T. Kjellstrom, Cadmium in the Environment, 2nd ed., CRC Press, Cleveland, Ohio, 1974. 5. H. H. Sanstead, in Effects and Dose-Response Relationships of Toxic Metals, G. F. Nordberg, ed., Elsevier, New York, 1976, pp. 511-526. 6. M. P. Verma, R. P. Sharma, and J. C. Street, Am. J. Vet. Res. 3 9, 1911 (1978). 7. R. P. Sharma and M. P. Verma, Am. J. Vet. Res. 41, 548 (1980). 8. K.J. Hardy and S. E. Bryan, in Biological Implications of Metals in the Environment, H. Drucher and R. E. Wildung, eds., US ERDA, Virginia , 1977, pp. 488-506. 9. W. G. Yasmineh and J. J. Yunis, Biochem. Biophys. Res. Commun. 35, 779 (1969). 10. W. D. Armstrong and C. W. Cart, Physical Chemistry, Laboratory Directions, Burgess Publishing, Minneapolis, Minnesota, 1963, pp. 112-113. 11. T. P. King and M. Spencer, J. Biol. Chem. 245, 6134 (1970). 12. T. R. Hughes, and I. M. Klotz, in Methods of Biochemical Analysis, D. Glick, ed., Interscience, New York, 1956, pp. 265-299. 13. K. Y. Chang and C. W. Carr, Biochem. Biophys. Acta. 157, 127 (1968). 14. G. Scatchard, Ann. N. Y. Acad. Sci. 51, 660 (1949). 15. M.S.M. Rao and H. Lal, J. Amer. Chem. Soc. 80, 3226 (1958). 16. J. H. R. Kagi and B. L. Vallee, J. Biol. Chem. 236, 2435 (1961). 17. P. Pulido, J. H. R. Kagi, and B. L. Vallee, Biochem. 5, 1768, (1966). 18. J. H. R. Kagi and B. L. Vallee, J. Biol. Chem. 235, 3460 (1960). 19. Y. Kojima, C. Berger, B. L. Vallee, and J. H. R. Kagi, Proc. Natl. Acad. Sci. USA 73, 3413 (1976). 20. H. R. Mahler and E. H. Cordes, Biological Chemistry, 2nd ed., Harper and Row, New York, 1971, p. 189. 21. T. Yamane and N. Davidson, J. Amer. Chem. Soc. 83, 2599 (1961). 22. G. L. Eichhorn, Advan. Chem. Ser. 62, 378 (1961). 23. Y. A. Shin and G. L. Eichhom, Biochemistry 7, 1026 (1968). 24. H. Richard, J. P. Schrieber, and M. Daune, Biopolymers 12, 1 (1973). 25. V. Mizuhira and M. Kimura, Proc. Elec. Microsc. Soc. Amer. 31,402 (1973).
