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The mobility of methylmercury in biological systems
B10~0RGAh?_CCBEM..~TRY
8,107-l
14 (1978)
107
The Mobility of Methylmercuryin BiologicalSystems
DALLAS L. PABENSTEIN and CHRISTOPHER A. EVAbiS Department of Chemistry. University of Alberta, Edmonton, Alberta, Canada
ABSTRACT Toxicology studies indicate that methylmercury in humans and other species is bonded to sulfbydryl ligands and that the methylmercury in such complexes is labile even though their thermodynamic stability is large. It is shown in this paper that bimolecular nucleophilic displacement of complexed ligand by sulfhydryl-deprotonated ligand is the major pathway for hgand exchange at physiological pH, while at the pH of the stomach the protonassisted dissociation of the complex is the predominant means by which exchange occurs. The dynamic and equilibrium aspects of the distribution of methylmercury between chloride and sulfhydryl ligands under the solution conditions of the stomach are also considered with respect to a possrble role for lipid-soluble CH$XgCl in the absorption of methylmercury from the StOlllXh-
INTRODUCTION Sulfhydryl ligands have a high affmity for mercury, and it generally is assumed that essentially all the methylmercury, CHaHg(II), in the blood and tissues of exposed animals or humans is complexed by sulfhydryl groups of cysteinecontaining peptides and proteins [l] . Yet, even though the thermodynamic stability of such complexes is high, the methylmercury must be labile, for it exchanges among the multitude of sulfhydryl groups it encounters, with some ultimately combining with target molecules in the brain_ The labile nature of methylmercury binding is also evidenced by the fact that sulfhydryl -containing chemotherapeutic agents, for example N-ace@-D,L-penicillamine [2] , extract methylmercury from the red blood cell and tissues and cause the total body burden to decrease more rapidly than by natural processes_ It is the purpose of this paper to account for the mobility of methylmercury in biological systems in terms of its solution chemistry. EXPERIMENTAL Proton magnetic resonance spectra were obtained on Varian A-60-D or HA-100 high resolution spectrometers at 25 + lo_ Chemical shifts were measured o Elsevier North-Holland, Inc., 1978
0006-3061-78-00084107$01.25
D. L. RABENSTEIN and C. A. EVANS
108
relative to tert-butyl alcohol, and are reported rektive to the methyl resonance of sodium 2,2dirnetbyl-2-silapentane-5-sulfonic acid (DSS). Positive shifts correspond to resonances of protons less shielded than those of DSS. An aqueous CH3HgOH stock solution was prepared by stirringCH3HgI with an excess of AgaO. The solution was standardized as described previously [3] _ Cysteine and giutathione (Nutritional Biochemicals Corp.) both were used as the free base. Solutions used in the nmr measurements were prepared in distilled, degassed water from the requisite amounts of the crystalline @and and the CHsHgOH standard solution under a nitrogen atmosphere to minimize oxidation_
RESULTS AND DI!3CUSSION The nuclear magnetic resonance results in Figs. 1 and 2 demonstrate that CHaHg(I1) exchanges rapidly among sulfhydryl ligands in mixtures. The proton
0.70 0.74 g
0.78
?z 0.82 i
0.86
=Ii
0.90
= 0.94 .U E Q 0.98 r u 1.02 1.06 1.10 I,
I,
1
I
3
I,
I
5
7
,
,
9
,
,
11
*
,
,
13
PH
FIG- 1. Chemical shift of the resonance for the CH3Hg(II) in aqueous solutions containing (A) 0.190 M methylmercury, (B) 0.150 M methylmercury and 0.130 M cysteine, (C) 0.150 M methylmercury and 0,150 M glutatbione ahd @) 0.100 M metbylmercury, cysteine and glutatbione. The data for solution (D) is indicated by open circIes_
MOBILITY OF METHYLMERCURY
IN BIOLOGICAL
SYSTEMS
109
260
252 246 236 228 220 212 0
.c =
z
v
204 196 188 180
172 164
-
C
LIx-l-J 1
3
5
7
9
11
13
PH
for CHaHg(II) FIG. 2. Jls~~~_ln given in the legend to Fig. 1.
iu aqueous solutions of the compositions
magnetic resonance spectrum of CHaHg(I1) consists of a singlet flanked symmetrically’ by two less intense satellite lines. The satellites are due to those methyl groups bonded to lg9Hg, which has a natural abundance of 16.9% and a nuclear spin of l/2, while the central resonance is due to methyl groups bonded to all other isotopes of mercury. The chemical shift of the resonance pattern is given by the position of the central resonance, the mercury-proton coupling constant by the separation of the satellites. In Fig. 1, the chemical shift of- the CHaHg(I1) in solutions containing (A) CHsHg(II), (B) equimolar concentrations of CHaHg(I1) and cysteine (C) equirnolar concentrations of CHaHg(I1) and glutathione and (D) equimolar concentrations of CHaHg(II), glutathione and cysteine is plotted as a function of coupling constants for the same solutions are plotted in PH. lgsHg-proton Fig. 2. The chemical shift of CHaHg(II) in the glutathione and cysteine complexes
110
D. L,. RABENSTEIN
and C. A. EVANS
is different. For the mixture, a single resonance pattern is observed at a chemical shift intermediate between those of the two complexes. A single resonance pattern, rather than separate resonance patterns for the individual complexes, indicates that the average lifetime of the individual compIexes is short on the nmr time scale (
CH,HgSR
kl
(1)
= CH,Hg+ + RSk-1
k,
CHaHgSR + H*
=+
CH,Hg+ + RSH
(2)
CHaHgSR
(3)
k-2 CHaHgSR + R’S-
2
+ RS-
If we use the CHaHg(II)-glutathione complex, CHsHgSG, as a model, we can identify those pathways responsible for the mobility of CHaHg(I1) in biological media. Rate constant kl is not known for CHaHgSG. However, from its formation constant of 1O15-s [S] and a diffusioncontrolled rate constant of lOlo m-l set-l for k_l. kl is predicted to be <10m6 set-s _If k_, is slower than diffusion controlled, kl will be even smaller, For CHaHgSG, kz = 600, k-2 = 5.1 X 109, and k3 =6X 108MB1 set-l [3]. The average number of exchanges of CHaHg(I1) between glutathione ligands per second is the sum of those via the individual pathways, as given by Equation 4.
A =kl + k2 [H+] 7
+ k3 [G!?]
(4)
MOBILITY
OF METHYLMERCURY
IN BIOLOGICAL
SYSTEMS
111
-5
-7
loa
[
GSH
1
frss
FIG. 3_ Logarithm of the average number of exchanges of CHsHg(II) among glutathione ligands per second by Reactions l-3 at pH 7 as a function of the concentration of free glutathione. The numbers correspond to the reactions.
where 7 represents the average lifetime of the glutathione complex and GS- the sulfhydryl-deprotonated form of glutathione. The magnitude of each of the terms on the right hand side of Equation 4 is plotted as a function of free glutathione concentration for pH 7 in Fig. 3. A value of 8.93 was used for the pK, of the sulfhydryl group of glutathione [6] in calculating the magnitude of the k3 [GS-] term_ The exchange rate shown for the first term corresponds to III = 10M6 set-I, the maximum possible value for kl, - the contribution of this term to the total exchange will be even less if kl is less. At pH 7, the amount of exchange by Reaction 2 is also negligibly smalI relative to exchange by Reaction 3. Even when the glutathione concentration is small, the CHaHg(I1) rapidly exchanges among the glutathione ligands by nucleophilic displacement of the complexed glutathione by sulfhydryl-deprotonated free glutathione. Rate parameters for the analogous reactions of CHaHg(I1) complexes of hemoglobin and other proteins are not known. However, the formation constants of such complexes are simiIar to that for CHaHgSG, so that by the same arguments as above kl is less than low6 set-l and Reaction 1 contributes negligibly to ligand exchange in such complexes. It also seems reasonable that k2 for protein complexes wiIl not be significantly larger than for CHaHgSG, in
112
D. L. RABENSTEIN
and C. A. EVANS
which cm Reaction 2 also contributes negligibly to ligand exchange at pH 7. These considerations, taken together with the known abundance of ~ulfhy&yl groups, indicate that Reaction 3 is the pathway by which CIWWI) exchanges among sulfhydryl ligands in biological media. For example, there is a 4800 to 8000 fold excess of sulfhydryl groups in blood at the blood CI-IaHg(II) levels where toxic symptoms first appear_ There is a similar excess of sulfhydryi groups over available CHaHg(I1) in other tissues [l] The existence of ghrtathione in all living cells, and the dependence of the extent to which CH,Hg(Ii) is taken up by red cells of rats on the intracellular glutathione level [7], suggests that the CHaHgSG complex plays a role in the mobility of CHaHg(II) in biological systems. The results in Fig. 3 indicate that at the ghttathione level of erythrocytes, typically 3 X 1W3 M [S] , CHaHg(II) exchange among glutathione ligands is rapid. Since the rate at which glutathione displaces CH3Hg(lI) from peptide and poteiu complexes is also probably fast, it seems reasonabIe to propose that displacement of CH3Hg(II) by glutathione and other small endogenous ligands according to Reaction 3 is largely responsible for the apparent lability of CH3Hg(II) in its thermodynamically-stable protein compIexes_ The ability of N-acetyl-D,L-penicillamine, a promising chemotherapeutic agent for methylmercury poisoning, to penetrate cell membranes and readily complex a substantial fraction of the intracellular CHaHg(I1) [2] indicates that displacement of biological ligands from CHaHg(I1) complexes by N-acetyl-D,L-penicillamine is also rapid. The above conclusions pertain to media of pH near 7. The dynamics of the CHaHg(II)-glutathione system, and presumably CH3Hg(II) complexes of other sulfhydryl-containing peptides and proteins, are different at the low pH of the stomach. The rate of exchange by Reaction 2 increases as the pH decreases, while at the same time the concentration of free glutathione in the sulfhydryldeprotonated form decreases causing the rate of exchange by Reaction 3 to decrease. The relative exchange rates by these two pathways (Fig_ 4) are such that, at the pH of the stomach, Reaction 2 is the predominant mechanism of CHaHg(I1) exchange among sulfhydryl ligands. In the stomach, CHaHg(I1) is in a medium of low pH (l-S-2.5) and high chloride concentration_ Under these conditions, a slzeable fraction of the methylmercury will be present as lipid-soluble CH,HgCl, For example, the distribution of CHaHg(i1) between the chloro and glutathione complexes is given as a function of the excess glutathione concentration in Table 1_Exchange-averaged mm spectra are observed for the CH3Hg(II) in pH 1.5 solutions containing both chloride and glutathione, indicating that the rate of interchange of CH3Hg(II) between the various Egands is fast on the nmr time scale. These results indicate that, if the lipid-soluble CHaHgCl is absorbed from the stomach into the blood stream [9], exchange of CHaHg(I1) among the ligands is sufficiently rapid to continually restore the equilibrium between the chloro and sulfhydryl com-
MOBILITY
OF METHYLMERCURY
IN BIOLOGICAL
SYSTEMS
113
FIG. 4. Lo,tithm of the average number of exchanges of CHaHg(II) among glutathione ligands per second by Reactions l-3 at pH 2 as a function of the concentration of free giutathione. The numbers correspond to the reactions.
plexes and thus convert the CHaHg(i1) to the absorbable form. The results shown in Table I for the distribution between the chloro and glutathione forms at pH 7.4 predict that onIy trace concentrations of CHaHgCl will be present in blood_ The rapid exchange of CHaHg(I1) among sulfhydryl ligands also has implications with respect to attempts to identify the molecules involved in the binding TABLE
1
Distribution of CH3Hg(II) Between Its Chloro and Glutathione Complexes as a Function of the Glutathione Concentration Percent of the CHaHg(I1) as CHaHgCl Free glutathione= concentration, IV 10-a 10-s 1o-4 1 o-a 10-a a Not complexed by CHaHg(I1). b [CT] = O.lOM. = [cl-] = 0.15M.
pH 2.0b 95.1 65.8 16.1 1.89 0.19
pH 7.4= 9.: 9.1 9.1 9.1 9.1
x x x x x
10-a lo-” 10-5 10-a 10-7
114
D. L. RABENSTEIN
and C_ A_ EVANS
of CH,Hg(II) at the subcellular level. Disruption of *the normal cellular and subcellular compartmentalization during analysis exposes the CH3Hg(II) to a multitude ofsuHhydryl-containingbiomolecules. Because bf the rapid exchange of CH3Hg(II) among sulfbydryl ligands, it seems likely that there will be a redistniution of CH3Hg(iI) among the ligands, making it difficult to identify the complexes origir~ally present by procedures which involve disruption of the cellular membranes_ This research WQS supported in part by grant jiom the National Research Council of Gvtada and by the University of Alberta. It is a pleasure to acknowledge the contributions of Dr. Mary Fatihurst to this work.
REFERENCES 1:
J_ T. MacGregor and T. W. Clarkson, in Adv. in Expt. Med. BioL. M. Friedman, ed. 48,64 (1974). 2. J_ Aaseth, Acta Pharmacol. et ToxicoL 39,289 (1976). 3. D. L. Rabenstein and M. T. Fairhurst,J. Amer. C’hem. SC. 97,2086 (1975)_ 4. Y. Takeda, T- Kunu& O_ Hoshino and T. Ukira, Toxicol. AppL PharmacoL 13, 165 (1968). 5. R. B. Simpson,J. Amer. Chem. Sot. 83,4711 (1961). 6. D. L. Rabenstein,J_ Amer. Chem. Sot. 95,2797 (1973). 7_ R_ Richardson and S. Murphy, Toxixol- AppL Pharmacoi 31.505 (1973). 8- F- k Abbott. in Uiiziurl Uzemticry. R. J. Henry, D. C. Cannon and J. W. Winkelman, ed., Harper and Row, New York, 1974, p_ 614. 9. H. J_ Se@ and J. M. Wood, Nature 248.456 (1974).
Received May 2. I977
8,107-l
14 (1978)
107
The Mobility of Methylmercuryin BiologicalSystems
DALLAS L. PABENSTEIN and CHRISTOPHER A. EVAbiS Department of Chemistry. University of Alberta, Edmonton, Alberta, Canada
ABSTRACT Toxicology studies indicate that methylmercury in humans and other species is bonded to sulfbydryl ligands and that the methylmercury in such complexes is labile even though their thermodynamic stability is large. It is shown in this paper that bimolecular nucleophilic displacement of complexed ligand by sulfhydryl-deprotonated ligand is the major pathway for hgand exchange at physiological pH, while at the pH of the stomach the protonassisted dissociation of the complex is the predominant means by which exchange occurs. The dynamic and equilibrium aspects of the distribution of methylmercury between chloride and sulfhydryl ligands under the solution conditions of the stomach are also considered with respect to a possrble role for lipid-soluble CH$XgCl in the absorption of methylmercury from the StOlllXh-
INTRODUCTION Sulfhydryl ligands have a high affmity for mercury, and it generally is assumed that essentially all the methylmercury, CHaHg(II), in the blood and tissues of exposed animals or humans is complexed by sulfhydryl groups of cysteinecontaining peptides and proteins [l] . Yet, even though the thermodynamic stability of such complexes is high, the methylmercury must be labile, for it exchanges among the multitude of sulfhydryl groups it encounters, with some ultimately combining with target molecules in the brain_ The labile nature of methylmercury binding is also evidenced by the fact that sulfhydryl -containing chemotherapeutic agents, for example N-ace@-D,L-penicillamine [2] , extract methylmercury from the red blood cell and tissues and cause the total body burden to decrease more rapidly than by natural processes_ It is the purpose of this paper to account for the mobility of methylmercury in biological systems in terms of its solution chemistry. EXPERIMENTAL Proton magnetic resonance spectra were obtained on Varian A-60-D or HA-100 high resolution spectrometers at 25 + lo_ Chemical shifts were measured o Elsevier North-Holland, Inc., 1978
0006-3061-78-00084107$01.25
D. L. RABENSTEIN and C. A. EVANS
108
relative to tert-butyl alcohol, and are reported rektive to the methyl resonance of sodium 2,2dirnetbyl-2-silapentane-5-sulfonic acid (DSS). Positive shifts correspond to resonances of protons less shielded than those of DSS. An aqueous CH3HgOH stock solution was prepared by stirringCH3HgI with an excess of AgaO. The solution was standardized as described previously [3] _ Cysteine and giutathione (Nutritional Biochemicals Corp.) both were used as the free base. Solutions used in the nmr measurements were prepared in distilled, degassed water from the requisite amounts of the crystalline @and and the CHsHgOH standard solution under a nitrogen atmosphere to minimize oxidation_
RESULTS AND DI!3CUSSION The nuclear magnetic resonance results in Figs. 1 and 2 demonstrate that CHaHg(I1) exchanges rapidly among sulfhydryl ligands in mixtures. The proton
0.70 0.74 g
0.78
?z 0.82 i
0.86
=Ii
0.90
= 0.94 .U E Q 0.98 r u 1.02 1.06 1.10 I,
I,
1
I
3
I,
I
5
7
,
,
9
,
,
11
*
,
,
13
PH
FIG- 1. Chemical shift of the resonance for the CH3Hg(II) in aqueous solutions containing (A) 0.190 M methylmercury, (B) 0.150 M methylmercury and 0.130 M cysteine, (C) 0.150 M methylmercury and 0,150 M glutatbione ahd @) 0.100 M metbylmercury, cysteine and glutatbione. The data for solution (D) is indicated by open circIes_
MOBILITY OF METHYLMERCURY
IN BIOLOGICAL
SYSTEMS
109
260
252 246 236 228 220 212 0
.c =
z
v
204 196 188 180
172 164
-
C
LIx-l-J 1
3
5
7
9
11
13
PH
for CHaHg(II) FIG. 2. Jls~~~_ln given in the legend to Fig. 1.
iu aqueous solutions of the compositions
magnetic resonance spectrum of CHaHg(I1) consists of a singlet flanked symmetrically’ by two less intense satellite lines. The satellites are due to those methyl groups bonded to lg9Hg, which has a natural abundance of 16.9% and a nuclear spin of l/2, while the central resonance is due to methyl groups bonded to all other isotopes of mercury. The chemical shift of the resonance pattern is given by the position of the central resonance, the mercury-proton coupling constant by the separation of the satellites. In Fig. 1, the chemical shift of- the CHaHg(I1) in solutions containing (A) CHsHg(II), (B) equimolar concentrations of CHaHg(I1) and cysteine (C) equirnolar concentrations of CHaHg(I1) and glutathione and (D) equimolar concentrations of CHaHg(II), glutathione and cysteine is plotted as a function of coupling constants for the same solutions are plotted in PH. lgsHg-proton Fig. 2. The chemical shift of CHaHg(II) in the glutathione and cysteine complexes
110
D. L,. RABENSTEIN
and C. A. EVANS
is different. For the mixture, a single resonance pattern is observed at a chemical shift intermediate between those of the two complexes. A single resonance pattern, rather than separate resonance patterns for the individual complexes, indicates that the average lifetime of the individual compIexes is short on the nmr time scale (
CH,HgSR
kl
(1)
= CH,Hg+ + RSk-1
k,
CHaHgSR + H*
=+
CH,Hg+ + RSH
(2)
CHaHgSR
(3)
k-2 CHaHgSR + R’S-
2
+ RS-
If we use the CHaHg(II)-glutathione complex, CHsHgSG, as a model, we can identify those pathways responsible for the mobility of CHaHg(I1) in biological media. Rate constant kl is not known for CHaHgSG. However, from its formation constant of 1O15-s [S] and a diffusioncontrolled rate constant of lOlo m-l set-l for k_l. kl is predicted to be <10m6 set-s _If k_, is slower than diffusion controlled, kl will be even smaller, For CHaHgSG, kz = 600, k-2 = 5.1 X 109, and k3 =6X 108MB1 set-l [3]. The average number of exchanges of CHaHg(I1) between glutathione ligands per second is the sum of those via the individual pathways, as given by Equation 4.
A =kl + k2 [H+] 7
+ k3 [G!?]
(4)
MOBILITY
OF METHYLMERCURY
IN BIOLOGICAL
SYSTEMS
111
-5
-7
loa
[
GSH
1
frss
FIG. 3_ Logarithm of the average number of exchanges of CHsHg(II) among glutathione ligands per second by Reactions l-3 at pH 7 as a function of the concentration of free glutathione. The numbers correspond to the reactions.
where 7 represents the average lifetime of the glutathione complex and GS- the sulfhydryl-deprotonated form of glutathione. The magnitude of each of the terms on the right hand side of Equation 4 is plotted as a function of free glutathione concentration for pH 7 in Fig. 3. A value of 8.93 was used for the pK, of the sulfhydryl group of glutathione [6] in calculating the magnitude of the k3 [GS-] term_ The exchange rate shown for the first term corresponds to III = 10M6 set-I, the maximum possible value for kl, - the contribution of this term to the total exchange will be even less if kl is less. At pH 7, the amount of exchange by Reaction 2 is also negligibly smalI relative to exchange by Reaction 3. Even when the glutathione concentration is small, the CHaHg(I1) rapidly exchanges among the glutathione ligands by nucleophilic displacement of the complexed glutathione by sulfhydryl-deprotonated free glutathione. Rate parameters for the analogous reactions of CHaHg(I1) complexes of hemoglobin and other proteins are not known. However, the formation constants of such complexes are simiIar to that for CHaHgSG, so that by the same arguments as above kl is less than low6 set-l and Reaction 1 contributes negligibly to ligand exchange in such complexes. It also seems reasonable that k2 for protein complexes wiIl not be significantly larger than for CHaHgSG, in
112
D. L. RABENSTEIN
and C. A. EVANS
which cm Reaction 2 also contributes negligibly to ligand exchange at pH 7. These considerations, taken together with the known abundance of ~ulfhy&yl groups, indicate that Reaction 3 is the pathway by which CIWWI) exchanges among sulfhydryl ligands in biological media. For example, there is a 4800 to 8000 fold excess of sulfhydryl groups in blood at the blood CI-IaHg(II) levels where toxic symptoms first appear_ There is a similar excess of sulfhydryi groups over available CHaHg(I1) in other tissues [l] The existence of ghrtathione in all living cells, and the dependence of the extent to which CH,Hg(Ii) is taken up by red cells of rats on the intracellular glutathione level [7], suggests that the CHaHgSG complex plays a role in the mobility of CHaHg(II) in biological systems. The results in Fig. 3 indicate that at the ghttathione level of erythrocytes, typically 3 X 1W3 M [S] , CHaHg(II) exchange among glutathione ligands is rapid. Since the rate at which glutathione displaces CH3Hg(lI) from peptide and poteiu complexes is also probably fast, it seems reasonabIe to propose that displacement of CH3Hg(II) by glutathione and other small endogenous ligands according to Reaction 3 is largely responsible for the apparent lability of CH3Hg(II) in its thermodynamically-stable protein compIexes_ The ability of N-acetyl-D,L-penicillamine, a promising chemotherapeutic agent for methylmercury poisoning, to penetrate cell membranes and readily complex a substantial fraction of the intracellular CHaHg(I1) [2] indicates that displacement of biological ligands from CHaHg(I1) complexes by N-acetyl-D,L-penicillamine is also rapid. The above conclusions pertain to media of pH near 7. The dynamics of the CHaHg(II)-glutathione system, and presumably CH3Hg(II) complexes of other sulfhydryl-containing peptides and proteins, are different at the low pH of the stomach. The rate of exchange by Reaction 2 increases as the pH decreases, while at the same time the concentration of free glutathione in the sulfhydryldeprotonated form decreases causing the rate of exchange by Reaction 3 to decrease. The relative exchange rates by these two pathways (Fig_ 4) are such that, at the pH of the stomach, Reaction 2 is the predominant mechanism of CHaHg(I1) exchange among sulfhydryl ligands. In the stomach, CHaHg(I1) is in a medium of low pH (l-S-2.5) and high chloride concentration_ Under these conditions, a slzeable fraction of the methylmercury will be present as lipid-soluble CH,HgCl, For example, the distribution of CHaHg(i1) between the chloro and glutathione complexes is given as a function of the excess glutathione concentration in Table 1_Exchange-averaged mm spectra are observed for the CH3Hg(II) in pH 1.5 solutions containing both chloride and glutathione, indicating that the rate of interchange of CH3Hg(II) between the various Egands is fast on the nmr time scale. These results indicate that, if the lipid-soluble CHaHgCl is absorbed from the stomach into the blood stream [9], exchange of CHaHg(I1) among the ligands is sufficiently rapid to continually restore the equilibrium between the chloro and sulfhydryl com-
MOBILITY
OF METHYLMERCURY
IN BIOLOGICAL
SYSTEMS
113
FIG. 4. Lo,tithm of the average number of exchanges of CHaHg(II) among glutathione ligands per second by Reactions l-3 at pH 2 as a function of the concentration of free giutathione. The numbers correspond to the reactions.
plexes and thus convert the CHaHg(i1) to the absorbable form. The results shown in Table I for the distribution between the chloro and glutathione forms at pH 7.4 predict that onIy trace concentrations of CHaHgCl will be present in blood_ The rapid exchange of CHaHg(I1) among sulfhydryl ligands also has implications with respect to attempts to identify the molecules involved in the binding TABLE
1
Distribution of CH3Hg(II) Between Its Chloro and Glutathione Complexes as a Function of the Glutathione Concentration Percent of the CHaHg(I1) as CHaHgCl Free glutathione= concentration, IV 10-a 10-s 1o-4 1 o-a 10-a a Not complexed by CHaHg(I1). b [CT] = O.lOM. = [cl-] = 0.15M.
pH 2.0b 95.1 65.8 16.1 1.89 0.19
pH 7.4= 9.: 9.1 9.1 9.1 9.1
x x x x x
10-a lo-” 10-5 10-a 10-7
114
D. L. RABENSTEIN
and C_ A_ EVANS
of CH,Hg(II) at the subcellular level. Disruption of *the normal cellular and subcellular compartmentalization during analysis exposes the CH3Hg(II) to a multitude ofsuHhydryl-containingbiomolecules. Because bf the rapid exchange of CH3Hg(II) among sulfbydryl ligands, it seems likely that there will be a redistniution of CH3Hg(iI) among the ligands, making it difficult to identify the complexes origir~ally present by procedures which involve disruption of the cellular membranes_ This research WQS supported in part by grant jiom the National Research Council of Gvtada and by the University of Alberta. It is a pleasure to acknowledge the contributions of Dr. Mary Fatihurst to this work.
REFERENCES 1:
J_ T. MacGregor and T. W. Clarkson, in Adv. in Expt. Med. BioL. M. Friedman, ed. 48,64 (1974). 2. J_ Aaseth, Acta Pharmacol. et ToxicoL 39,289 (1976). 3. D. L. Rabenstein and M. T. Fairhurst,J. Amer. C’hem. SC. 97,2086 (1975)_ 4. Y. Takeda, T- Kunu& O_ Hoshino and T. Ukira, Toxicol. AppL PharmacoL 13, 165 (1968). 5. R. B. Simpson,J. Amer. Chem. Sot. 83,4711 (1961). 6. D. L. Rabenstein,J_ Amer. Chem. Sot. 95,2797 (1973). 7_ R_ Richardson and S. Murphy, Toxixol- AppL Pharmacoi 31.505 (1973). 8- F- k Abbott. in Uiiziurl Uzemticry. R. J. Henry, D. C. Cannon and J. W. Winkelman, ed., Harper and Row, New York, 1974, p_ 614. 9. H. J_ Se@ and J. M. Wood, Nature 248.456 (1974).
Received May 2. I977