Arch. Environ. Contain, Toxicol. 23,426-430 (1992)
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Structural Changes of Proteins in Fish Red Blood Cells after Copper and Mercury Treatment Krzysztof Gwozdzinski Department of Biophysics, University of Lodz, 90-237 Lodz, Poland
Abstract. Exposure of fish red blood cells to increased concentrations (0.05-0.3 mmol/L) of copper and mercury ions may initiate structural changes in cells as detected by spin labeling method. Both heavy metals decreased membrane fluidity as indicated by methyl 5-doxylpalmitate and methyl 12-doxylstearate spectra. Furthermore, copper and mercury have been found to induce conformational alterations of internal peptides and proteins as determined by using 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl. Both heavy metal ions changed the internal viscosity of red blood cells. These results suggest that the possible cause of the damage of cells may be metal-protein interactions in the cells, but may exclude the oxidative mechanism of such damage.
Copper and mercury are strongly toxic metal ions which cause the destruction of red blood cells. Both heavy metal ions induce hemolysis of human (Piriou et al. 1987; Ichikawa et al. 1987) and fish erythrocytes (Gwozdzinski et al. 1992 in press). Copper and mercury lower membrane permeability for non-electrolytes in human erythrocytes and carp erythrocytes (Yaeger et al. 1979; Gwozdzinski 1985). Both heavy metals decrease intracellular glutathione levels (Ribarov and Benov 1981 ; Piriou et al. 1987). Copper and mercury ions are capable of catalyzing peroxidation in liposomes and erythrocyte membranes (Ribarov and Benov 1981; C h a n e t al. 1982). Fish erythrocytes are characterized by essential differences in structure and function when compared to mammalian erythrocytes. For example, fish erythrocytes contain higher levels of phospholipids in their plasma membranes, and the degree of phospholipid saturation is considerably lower (Bolls and Fange 1979). In contrast to mammalian erytrocytes, fish erythrocytes do not transport D-glucose (Bolls et al. 1971). Recently, we have shown that fish erythrocytes are more fragile and more sensitive to copper-induced hemolysis than human erythrocytes (Gwozdzinski et al. 1992 in press). The aim of this study was to elucidate the action of copper and mercury on the structure of fish red blood cells using spin label methods.
Materials and Methods The adult carp (Cyprinus carpio L.) were adapted to aquarium conditions for two weeks at temperatures of 10 -+ 2°C. Blood was drawn by tail vein puncture into a heparinized syringe. After centrifugation (3 min at 1500 x g) erythrocytes were washed twice with 0.6% NaCI. Methyl 5-doxylpalmitate, methyl 12-doxylstearate, maleimide spin label (MSL, 4-maleimido-2,2,6,6-tetramethylpiperidine-l-oxyl)and Tempone (4-oxo-2,2,6,6-tetramethylpiperidine-l-oxyl) were synthesized as described in detail elsewhere (Gwozdzinski 1991a). Erythrocytes were labeled by the introduction of aliquots of the ethanol solution of spin label fatty acid esters. The final ethanol concentration in erythrocyte suspension was less than 0.05% (v/v). After 30 rain incubation at room temperature, erythrocytes were washed with 0.6% NaCI. In the case of MSL, erythrocytes were incubated for I h at room temperature, and then the unbound spin label was removed by several washings until the ESR signal in supernatant disappeared. After labeling, erythrocyte suspensions were incubated at room temperature for 1 h with increasing concentrations (0.05, 0.1 and 0.3 retool/L) of copper sulfate or mercury chloride. Intracellular viscosity of red blood cells was determined according to Morse (1986). The red blood cells were incubated for 1 h with Ternpone spin label. Before measurement, erytbrocytes were washed with 80 mmol/L potassium ferricyanide, which was applied as a broadening agent in order to eliminate the extracellular signal of the spin label. ESR measurements were performed at room temperature, using SE/X-20 (X-band) spectrometer (Wroclaw Technical University, Poland). Statistical analyses included the calculation of means +- S.D. The significance of differences was estimated by Tukey's test for multiple comparisons.
Results and Discussion Figure 1 shows typical spectra of methyl 5-doxylpalmitate (a) and methyl 12-doxylstearate (b) incorporated into fish red blood cell membranes. The experimental parameter h+j/ho, where h+l and h o are the heights of low-field line and middle line of the spectra respectively, was determined as a semiquantitative measure of acyl chain flexibility corresponding to lipid bilayer fluidity (Morrisett et al. 1975). Figures 2 and 3 show the effects of increasing concentrations of copper and mercury ions on lipid bilayer dynamics. These results indicate that both heavy metals induce significant increase of the h+ ~/ho parameter. This increse is significant (approx. 5%) for methyl 12-
Effects of Copper and Mercury on Fish Blood Cells
427
resultant immobilization of the label may result from the immobilizing effect of heavy metal ions on both internal fluid and membrane proteins. The results presented herein suggest the possible changes in protein conformation of intracellular peptides and/or proteins. The intracellular viscosity of cells was also determined by the comparison of the motion of spin label from the inside of cells with the motion of spin label in water. Accordingly the internal viscosity of erythrocytes was evaluated from the ratio:
/
Tc (erythrocyte)/'rc (water) m: qq(erythrocyte)/q0(water )
a where % are rotational correlation times for Tempone in erythrocytes and in a bulk of water, respectively, and ~1 are internal viscosity of erythrocytes and water (Morse 1986). The rotational correlation time was calculated from the equation: % = k * W o * [(ho/h_1) j/2 - 1]
h+l
H
Fig. 1. ESR spectra of methyl 5-doxylpalmitate (a) and methyl 12doxylstearate (b) incorporated into carp red blood cells. The "motion parameter" h+Jho was calculated, where h+l and ho are heights of low-field line and middle-field line, respectively
doxylstearate, in which the nitroxide reporting group is located deeper, and these changes are relevant to the decrease of membrane lipid fluidity. Figure 4 shows the spectrum of maleimide spin label attached to fish red blood ceils. Under neutral pH, maleimide reacts mainly with thiol groups of proteins (Berliner 1983). Maleimide spin label attached to intact human erythrocytes yields a triplet spectrum, whereas the spectrum of the label bound to fish red blood cells reveals the ESR signals of weakly immobilized (narrow-line, hw) and strongly immobilized (broad-line, h0 components. The analyses of the spectra of maleimide attached to red blood cells was performed by the calculation of hw/hs ratio, which is a very sensitive measure of the physical state of proteins in membrane (Fung 1983). Small perturbations in the nearest proximity of the label bound to membrane proteins may produce significant changes in the MSL spectra used for the monitoring of conformational changes of proteins. Figures 2 and 3 show the effects of various concentrations of copper and mercury on internal protein mobility. Recently, we have reported that MSL penetrates erythrocyte membrane and reacts faster with internal peptides and/or proteins than with membrane proteins (Gwozdzinski 1991 b). We also showed that more than 70% of label was bound with intracellular fluid. The
where k is a constant equal to 6.5 * 10 -~° for nitroxide spin label, W o is the width of midfield line derivative, ha is the height of the midfield line derivative, and h_ ~ is the height of high-field line derivative. The effect of copper and mercury on the internal viscosity of human and fish erythrocytes is shown in Tables 1 and 2. The most pronounced effects of copper and mercury occurred at the concentration of 0.1 mmol/L for both metals. Moreover, both heavy metals increased the internal viscosity of fish red blood cells while, in contrast, they decreased the internal viscosity of human red blood cells. Copper and mercury initiate hemolysis of erythrocytes but the detailed mechanism of this process is unclear. Ribarov and Benov (1981) reported that the possible cause of hemolysis of red blood cells may be peroxidation of membrane lipids. On the other hand, Piriou et al. (1987) and Ichikawa et al. (1987) did not find lipid peroxidation products in copper- and/or mercurytreated erythrocytes. Hence, they suggested that lipid peroxidation is not the cause of erythrocyte hemolysis. Copper ions also catalyzed lipid peroxidation in liposomes and erythrocyte membranes (Chart et al. 1982). The decrease of the motion parameter h+~/ho of two spin label fatty acids located at different depths within the lipid bilayer indicates the lowering of membrane fluidity in copper- or mercury-treated erythrocytes. These results are in agreement with the observation that lipid peroxidation produces the decrease of liposome fluidity (Bartosz et al. 1987). Nevertheless, we were unable to find lipid peroxidation products (TBARS - thiobarbituric acid reactive substances) in copper-treated erythrocytes (Gabryelak and Gwozdzinski, 1987). The decrease of membrane fluidity is a consequence of lipid-protein interaction rather than the lipid peroxidation process. The decrease of membrane fluidity is in good agreement with data reported earlier for human erythrocytes (Gwozdzinski 1991a). Recently, we have shown that radiation, e . g . , water radiolysis products, are the cause of the increase in membrane fluidity and the increase of thiobarbituric acid reacting substances (Gwozdzinski 1991 b). Copper oxidizes thiol groups of proteins and thus leads to the formation of-S-S- bridges (Salhany et al. 1978). Salhany et al. (1978) found that copper ions diminished the amounts of band 1 + 2 (spectrin), band 4.2 and band 5 (actin), and produced high molecular weight aggregates. They also showed that band 3 was located at its dirneric position on the gel. Mercury ions are capable of binding to band 3 (Ralston and Crisp 1981).
428
K. Gwozdzinski
%
240 200 ~
}:ii!? ~ i'i!:::i:} I
~ • :i! ~ :~, .:i:
160
Fig. 2. Percent changes in "motion parameter" h+ ~/h0 of methyl 5-doxylpalmitate and methyl 12-doxylstearate and h,Jh~ ratio for maleimide spin label with respect to control values as a function of copper concentration. Met 5-DP (control: 0.547 -+ 0.012) no significant changes (n = 4); Met t2-DS (control: 0.654 +- 0.007), control vs. 0.1 mmol/L p < 0.005, control vs. 0.3 mmol/L p < 0.005 (n = 4); MSL (control: 4.86 -+ 1.05), control vs. 0.05 mmol/L n.s., control vs. 0.1 mmol/L p < 0.01, control vs. 0.3 mmol/L p < 0.005 (n = 6).
120 80 40 :::::::.:
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Mercury disrupts the interaction of cytoskeleton proteins with the membrane. Furthermore, solubilization of fish erythrocyte membranes was observed when fish were exposed to mercury (Panigrahi and Misra 1979). Copper ions generate superoxide radical in erythrocyte membranes which seems to underlie the oxidation of -SH groups (Kumar et al. 1978). Superoxide radicals can be also generated during copper-dependent autoxidation of hemoglobin to methemoglobin (Rifkind 1974). Superoxide radicals and hydrogen peroxide, in the presence of traces of heavy metals, can promote the generation of hydroxyl radicals (in the Haber-Weiss reaction) or other oxygen species of equivalent reactivity (Rowley and Halliwell 1983). Interestingly, we also found that copper and mercury did not inactivate antioxidant enzymes such as superoxide dismutase, catalase, peroxidase and glutathione peroxidase (Gwozdzinski et aI. 1992, in press).
MSL
::.: ,
Fig. 3. Percent changes in "motion parameter" h+ l/ho of methyl 5-doxylpalmitate and methyl 12-doxylstearate and hw/h.~ratio for maleimide spin label with respect to control values as a function of mercury concentration. Met 5-DP (control: 0.547 -+ 0.012)control vs. 0.1 mmol/L p < 0.05, control vs. 0.3 mmol/L p < 0.05 (n = 4); Met I2-DS (control: 0.654 -+ 0.007) control vs. 0.1 mmot/L n.s., control vs. 0.3 mmol/L p < 0.001 (n = 4); MSL (control: 4.86 --- 1.05) control vs. 0.05 mmol/L n.s., control vs. 0.1 mmol/L n.s., control vs. 0.3 mmol/L p < 0.005 (n = 6).
Using maleimide spin label, we observed increase in hw/h s ratio, which reflects the copper- and mercury-induced changes in the protein conformation or the disposition of spin labeled fragments of proteins. Both heavy metals exhibited specific action on internal viscosity in carp erythocytes at the concentration of 0.1 mmol/L. It seems that the observed alterations may be related to the formation of -S-S- bridges in the case of Cu 2+ and -S-Hg-S- bridges in the case of Hg 2+ within some molecules of peptides and proteins as well as between some neighbouring molecules of proteins. In the case of mercury it appears that also membrane proteins at the cytosolic site may be affected after the disruption of cytoskeleton. This mechanism of action of both heavy metals on cell damage does not explain why internal viscosity increases in human erythrocytes and decreases in carp erythrocytes. But it has to be remembered that carp erythrocytes are more complex cells than human erythro-
Effects of Copper and Mercury on Fish Blood Cells
429
r
bw
H 1,
Fig. 4. ESR spectrum of 4-maleimido-2,2,6,6-tetramethylpiperidine- 1oxyl attached to carp red blood cells. The ratio hw/h s was calculated. The amplitudes of spin label bound to strongly and weakly immobilized binding sites are indicated by h w and h~, respectively.
Table 1. Effect of copper ions on internal viscosity of human and carp red blood cells
Table 2. Effect of mercury ions on internal viscosity of human and red blood cells
Copper concentration (retool/L)
Human rbc Carp rbc
Mercury concentration (retool/L)
0
0.05
0. l
0.3
4.43 -+ 0.13 3.01 -+ 0.23
4.35 -+ 0.25 2.96 -+ 0.19
4.02 a +- 0.24 3.22 -+ 0.22
4.25 -+ 0.07 2.98 -+ 0.15
aControl vs. 0.1 mmol/L p < 0.025
cytes (the presence of nuclei, microfilaments connected to cellular m e m b r a n e , huge variety of m e m b r a n e proteins, higher content of polyunsaturated fatty acids (Bolis and Fange 1979), inability to transport D-glucose (Bolis et al. 1971)). The mechanism in which -SH groups of protein take part seems to be in agreement with the report by Barnes and Frieden, who showed that -SH blockers inhibited Cu 2+ -induced hemolysis. In conclusion, it emerges from this study that the action of oxygen radical species in the damage of cells is probably less important and the major cause of cell injury m a y be the alterations in protein structure.
Human rbc Carp rbc
0
0.05
0. I
0.3
4.43 -+ 0.13 3.01 -+ 0.23
4.36 -+ 0.13 2.99 -+ 0.28
4.07 a -+ 0.22 3.25 -+ 0.21
4.35 -+ 0.17 2.96 -+ 0.20
aControl vs 0.1 mmol/L p < 0.025
References Bartosz G, Christ G, Basse H, Stephan R and Gartner H (1987) An ESR study of the lipid peroxidation of lecitin multilayers. Z Naturfosch 43a: 1381-1384 Berliner LJ (1983) The spin label approach to labeling membrane protein sulfhydryl groups. Ann NY Acad Sci 414:153-161 Bolis LP and Fange R (1979) Lipid composition of erythrocyte membrane of some marine fish. Comp Biochem Physio162b:345-348 Bolis LP, Luly P and B aroncelli V (1971 ) D(+)glucose permeability in brown trout Salmo trutta L. erythrocytes. J Fish Biol 3:373-375
430
Chan PC, Palmer OG and Kesner L (1982) Copper (II)-catalyzed lipid peroxidation in liposomes and erythrocyte membrane. Lipids 17:331-337 Fung LW (1983) Analysis of spin labeled erythrocyte membrane. Ann NY Acad Sci 414:162-167 Gabryelak T, Gwozdzinski K (1987) The effect of paraquat and cupric ions on superoxide dismutase and glutathione peroxidase activities in carp erythrocytes. Stud Kieleckie 4:109-I 17 Gwozdzinski K (1985) Effect of cupric ions on the permeability of erythrocyte membrane to non-electrolyte spin labels. Physiol Chem Phys Med NMR 17:431-434 Gwozdzinski K (1991a) A spin label study of the action of cupric and mercuric ions on human red blood cells. Toxicology 65:315-323 --(1991b) Radiation-induced structural alterations in fish red blooc cells. Free Radical Biol Med 11:557-561 [chikawa H, Ronowicz K, Hicks M, Gebicki J (1987) Lipid peroxidation is not the cause of lysis of human erythrocytes exposed to inorganic or methylmercury. Arch Biochem Biophys 259:46-51 Kumar KS, Rowse C, Hochstein P (1978) Copper induced generation of superoxide in human red blood cell membrane. Biochem Biophys Res Commun 83:587-592 Morrisett JD, Pownall HJ, Plumlee RT, Smith LC, Zahner ZE, Esfahani M, Wakil SJ (1975) Multiple thermotropic phase transition in Escherichia coli membranes and lipids, J Biol Chem 250:69696976 Morse PD (1986) Determining intracellular viscosity from rotational motion of spin labels. Methods Enzymol 127:239-249
K, Gwozdzinski
Panigrahi AK, Misra BN (1979) Effect of mercury on the morphology of erythrocytes in Anabas scanders. Bull Environ Contain Toxicol 23:784-787 Piriou A, Tallineau C, Chahboun S, Pontcharraud R, Guillard O (1987) Copper-induced lipid peroxidation and hemolysis in whole blood evidence for lack correlation. Toxicology 47:351-361 Ralston GB, Crisp EA (]981) The action of organic mercurials on the erythrocyte membrane. B iochem B iophys Acta 649:98-104 Ribarov S, Benov L ( 1981 ) Relationship between the hemolysis action of heavy metals and lipid peroxidation. Biochim Biophys Acta 640:721-726 Rifkind JM (1974) Copper and the autoxidation of hemoglobin. Biochemistry 13:2475-2481 Rowley DA, Halliwell B (1983) Superoxide-dependent and ascorbatedependent formation of hydroxyl radicals in the presence of copper salts: A physiologically significant reaction ? Arch Biochem Biophys 225:279-284 Salhany JM, Swanson JC, Cordes KA, Gaines SB, Gaines KC (1978) Evidence suggesting direct oxidation of human erythrocyte membrane sulfhydryls by copper. Biochem Biophys Res Commun 82:1294-1299 Yaeger Y, Nathan 1, Dvilansky A, Meyerstein N (1979) Permeability of fresh and stored human erythrocytes to glycerol and its acylated derivatives. Experientia 35:1673-1674 Manuscript received February 18, 1992 and in revised form June 26, 1992.
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E nvironmental
contam ination and Ioxicology
© 1992 Springer-Verlag New York Inc.
Structural Changes of Proteins in Fish Red Blood Cells after Copper and Mercury Treatment Krzysztof Gwozdzinski Department of Biophysics, University of Lodz, 90-237 Lodz, Poland
Abstract. Exposure of fish red blood cells to increased concentrations (0.05-0.3 mmol/L) of copper and mercury ions may initiate structural changes in cells as detected by spin labeling method. Both heavy metals decreased membrane fluidity as indicated by methyl 5-doxylpalmitate and methyl 12-doxylstearate spectra. Furthermore, copper and mercury have been found to induce conformational alterations of internal peptides and proteins as determined by using 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl. Both heavy metal ions changed the internal viscosity of red blood cells. These results suggest that the possible cause of the damage of cells may be metal-protein interactions in the cells, but may exclude the oxidative mechanism of such damage.
Copper and mercury are strongly toxic metal ions which cause the destruction of red blood cells. Both heavy metal ions induce hemolysis of human (Piriou et al. 1987; Ichikawa et al. 1987) and fish erythrocytes (Gwozdzinski et al. 1992 in press). Copper and mercury lower membrane permeability for non-electrolytes in human erythrocytes and carp erythrocytes (Yaeger et al. 1979; Gwozdzinski 1985). Both heavy metals decrease intracellular glutathione levels (Ribarov and Benov 1981 ; Piriou et al. 1987). Copper and mercury ions are capable of catalyzing peroxidation in liposomes and erythrocyte membranes (Ribarov and Benov 1981; C h a n e t al. 1982). Fish erythrocytes are characterized by essential differences in structure and function when compared to mammalian erythrocytes. For example, fish erythrocytes contain higher levels of phospholipids in their plasma membranes, and the degree of phospholipid saturation is considerably lower (Bolls and Fange 1979). In contrast to mammalian erytrocytes, fish erythrocytes do not transport D-glucose (Bolls et al. 1971). Recently, we have shown that fish erythrocytes are more fragile and more sensitive to copper-induced hemolysis than human erythrocytes (Gwozdzinski et al. 1992 in press). The aim of this study was to elucidate the action of copper and mercury on the structure of fish red blood cells using spin label methods.
Materials and Methods The adult carp (Cyprinus carpio L.) were adapted to aquarium conditions for two weeks at temperatures of 10 -+ 2°C. Blood was drawn by tail vein puncture into a heparinized syringe. After centrifugation (3 min at 1500 x g) erythrocytes were washed twice with 0.6% NaCI. Methyl 5-doxylpalmitate, methyl 12-doxylstearate, maleimide spin label (MSL, 4-maleimido-2,2,6,6-tetramethylpiperidine-l-oxyl)and Tempone (4-oxo-2,2,6,6-tetramethylpiperidine-l-oxyl) were synthesized as described in detail elsewhere (Gwozdzinski 1991a). Erythrocytes were labeled by the introduction of aliquots of the ethanol solution of spin label fatty acid esters. The final ethanol concentration in erythrocyte suspension was less than 0.05% (v/v). After 30 rain incubation at room temperature, erythrocytes were washed with 0.6% NaCI. In the case of MSL, erythrocytes were incubated for I h at room temperature, and then the unbound spin label was removed by several washings until the ESR signal in supernatant disappeared. After labeling, erythrocyte suspensions were incubated at room temperature for 1 h with increasing concentrations (0.05, 0.1 and 0.3 retool/L) of copper sulfate or mercury chloride. Intracellular viscosity of red blood cells was determined according to Morse (1986). The red blood cells were incubated for 1 h with Ternpone spin label. Before measurement, erytbrocytes were washed with 80 mmol/L potassium ferricyanide, which was applied as a broadening agent in order to eliminate the extracellular signal of the spin label. ESR measurements were performed at room temperature, using SE/X-20 (X-band) spectrometer (Wroclaw Technical University, Poland). Statistical analyses included the calculation of means +- S.D. The significance of differences was estimated by Tukey's test for multiple comparisons.
Results and Discussion Figure 1 shows typical spectra of methyl 5-doxylpalmitate (a) and methyl 12-doxylstearate (b) incorporated into fish red blood cell membranes. The experimental parameter h+j/ho, where h+l and h o are the heights of low-field line and middle line of the spectra respectively, was determined as a semiquantitative measure of acyl chain flexibility corresponding to lipid bilayer fluidity (Morrisett et al. 1975). Figures 2 and 3 show the effects of increasing concentrations of copper and mercury ions on lipid bilayer dynamics. These results indicate that both heavy metals induce significant increase of the h+ ~/ho parameter. This increse is significant (approx. 5%) for methyl 12-
Effects of Copper and Mercury on Fish Blood Cells
427
resultant immobilization of the label may result from the immobilizing effect of heavy metal ions on both internal fluid and membrane proteins. The results presented herein suggest the possible changes in protein conformation of intracellular peptides and/or proteins. The intracellular viscosity of cells was also determined by the comparison of the motion of spin label from the inside of cells with the motion of spin label in water. Accordingly the internal viscosity of erythrocytes was evaluated from the ratio:
/
Tc (erythrocyte)/'rc (water) m: qq(erythrocyte)/q0(water )
a where % are rotational correlation times for Tempone in erythrocytes and in a bulk of water, respectively, and ~1 are internal viscosity of erythrocytes and water (Morse 1986). The rotational correlation time was calculated from the equation: % = k * W o * [(ho/h_1) j/2 - 1]
h+l
H
Fig. 1. ESR spectra of methyl 5-doxylpalmitate (a) and methyl 12doxylstearate (b) incorporated into carp red blood cells. The "motion parameter" h+Jho was calculated, where h+l and ho are heights of low-field line and middle-field line, respectively
doxylstearate, in which the nitroxide reporting group is located deeper, and these changes are relevant to the decrease of membrane lipid fluidity. Figure 4 shows the spectrum of maleimide spin label attached to fish red blood ceils. Under neutral pH, maleimide reacts mainly with thiol groups of proteins (Berliner 1983). Maleimide spin label attached to intact human erythrocytes yields a triplet spectrum, whereas the spectrum of the label bound to fish red blood cells reveals the ESR signals of weakly immobilized (narrow-line, hw) and strongly immobilized (broad-line, h0 components. The analyses of the spectra of maleimide attached to red blood cells was performed by the calculation of hw/hs ratio, which is a very sensitive measure of the physical state of proteins in membrane (Fung 1983). Small perturbations in the nearest proximity of the label bound to membrane proteins may produce significant changes in the MSL spectra used for the monitoring of conformational changes of proteins. Figures 2 and 3 show the effects of various concentrations of copper and mercury on internal protein mobility. Recently, we have reported that MSL penetrates erythrocyte membrane and reacts faster with internal peptides and/or proteins than with membrane proteins (Gwozdzinski 1991 b). We also showed that more than 70% of label was bound with intracellular fluid. The
where k is a constant equal to 6.5 * 10 -~° for nitroxide spin label, W o is the width of midfield line derivative, ha is the height of the midfield line derivative, and h_ ~ is the height of high-field line derivative. The effect of copper and mercury on the internal viscosity of human and fish erythrocytes is shown in Tables 1 and 2. The most pronounced effects of copper and mercury occurred at the concentration of 0.1 mmol/L for both metals. Moreover, both heavy metals increased the internal viscosity of fish red blood cells while, in contrast, they decreased the internal viscosity of human red blood cells. Copper and mercury initiate hemolysis of erythrocytes but the detailed mechanism of this process is unclear. Ribarov and Benov (1981) reported that the possible cause of hemolysis of red blood cells may be peroxidation of membrane lipids. On the other hand, Piriou et al. (1987) and Ichikawa et al. (1987) did not find lipid peroxidation products in copper- and/or mercurytreated erythrocytes. Hence, they suggested that lipid peroxidation is not the cause of erythrocyte hemolysis. Copper ions also catalyzed lipid peroxidation in liposomes and erythrocyte membranes (Chart et al. 1982). The decrease of the motion parameter h+~/ho of two spin label fatty acids located at different depths within the lipid bilayer indicates the lowering of membrane fluidity in copper- or mercury-treated erythrocytes. These results are in agreement with the observation that lipid peroxidation produces the decrease of liposome fluidity (Bartosz et al. 1987). Nevertheless, we were unable to find lipid peroxidation products (TBARS - thiobarbituric acid reactive substances) in copper-treated erythrocytes (Gabryelak and Gwozdzinski, 1987). The decrease of membrane fluidity is a consequence of lipid-protein interaction rather than the lipid peroxidation process. The decrease of membrane fluidity is in good agreement with data reported earlier for human erythrocytes (Gwozdzinski 1991a). Recently, we have shown that radiation, e . g . , water radiolysis products, are the cause of the increase in membrane fluidity and the increase of thiobarbituric acid reacting substances (Gwozdzinski 1991 b). Copper oxidizes thiol groups of proteins and thus leads to the formation of-S-S- bridges (Salhany et al. 1978). Salhany et al. (1978) found that copper ions diminished the amounts of band 1 + 2 (spectrin), band 4.2 and band 5 (actin), and produced high molecular weight aggregates. They also showed that band 3 was located at its dirneric position on the gel. Mercury ions are capable of binding to band 3 (Ralston and Crisp 1981).
428
K. Gwozdzinski
%
240 200 ~
}:ii!? ~ i'i!:::i:} I
~ • :i! ~ :~, .:i:
160
Fig. 2. Percent changes in "motion parameter" h+ ~/h0 of methyl 5-doxylpalmitate and methyl 12-doxylstearate and h,Jh~ ratio for maleimide spin label with respect to control values as a function of copper concentration. Met 5-DP (control: 0.547 -+ 0.012) no significant changes (n = 4); Met t2-DS (control: 0.654 +- 0.007), control vs. 0.1 mmol/L p < 0.005, control vs. 0.3 mmol/L p < 0.005 (n = 4); MSL (control: 4.86 -+ 1.05), control vs. 0.05 mmol/L n.s., control vs. 0.1 mmol/L p < 0.01, control vs. 0.3 mmol/L p < 0.005 (n = 6).
120 80 40 :::::::.:
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0,1
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copper concentration (mmol/L) 5-DP
~
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Mercury disrupts the interaction of cytoskeleton proteins with the membrane. Furthermore, solubilization of fish erythrocyte membranes was observed when fish were exposed to mercury (Panigrahi and Misra 1979). Copper ions generate superoxide radical in erythrocyte membranes which seems to underlie the oxidation of -SH groups (Kumar et al. 1978). Superoxide radicals can be also generated during copper-dependent autoxidation of hemoglobin to methemoglobin (Rifkind 1974). Superoxide radicals and hydrogen peroxide, in the presence of traces of heavy metals, can promote the generation of hydroxyl radicals (in the Haber-Weiss reaction) or other oxygen species of equivalent reactivity (Rowley and Halliwell 1983). Interestingly, we also found that copper and mercury did not inactivate antioxidant enzymes such as superoxide dismutase, catalase, peroxidase and glutathione peroxidase (Gwozdzinski et aI. 1992, in press).
MSL
::.: ,
Fig. 3. Percent changes in "motion parameter" h+ l/ho of methyl 5-doxylpalmitate and methyl 12-doxylstearate and hw/h.~ratio for maleimide spin label with respect to control values as a function of mercury concentration. Met 5-DP (control: 0.547 -+ 0.012)control vs. 0.1 mmol/L p < 0.05, control vs. 0.3 mmol/L p < 0.05 (n = 4); Met I2-DS (control: 0.654 -+ 0.007) control vs. 0.1 mmot/L n.s., control vs. 0.3 mmol/L p < 0.001 (n = 4); MSL (control: 4.86 --- 1.05) control vs. 0.05 mmol/L n.s., control vs. 0.1 mmol/L n.s., control vs. 0.3 mmol/L p < 0.005 (n = 6).
Using maleimide spin label, we observed increase in hw/h s ratio, which reflects the copper- and mercury-induced changes in the protein conformation or the disposition of spin labeled fragments of proteins. Both heavy metals exhibited specific action on internal viscosity in carp erythocytes at the concentration of 0.1 mmol/L. It seems that the observed alterations may be related to the formation of -S-S- bridges in the case of Cu 2+ and -S-Hg-S- bridges in the case of Hg 2+ within some molecules of peptides and proteins as well as between some neighbouring molecules of proteins. In the case of mercury it appears that also membrane proteins at the cytosolic site may be affected after the disruption of cytoskeleton. This mechanism of action of both heavy metals on cell damage does not explain why internal viscosity increases in human erythrocytes and decreases in carp erythrocytes. But it has to be remembered that carp erythrocytes are more complex cells than human erythro-
Effects of Copper and Mercury on Fish Blood Cells
429
r
bw
H 1,
Fig. 4. ESR spectrum of 4-maleimido-2,2,6,6-tetramethylpiperidine- 1oxyl attached to carp red blood cells. The ratio hw/h s was calculated. The amplitudes of spin label bound to strongly and weakly immobilized binding sites are indicated by h w and h~, respectively.
Table 1. Effect of copper ions on internal viscosity of human and carp red blood cells
Table 2. Effect of mercury ions on internal viscosity of human and red blood cells
Copper concentration (retool/L)
Human rbc Carp rbc
Mercury concentration (retool/L)
0
0.05
0. l
0.3
4.43 -+ 0.13 3.01 -+ 0.23
4.35 -+ 0.25 2.96 -+ 0.19
4.02 a +- 0.24 3.22 -+ 0.22
4.25 -+ 0.07 2.98 -+ 0.15
aControl vs. 0.1 mmol/L p < 0.025
cytes (the presence of nuclei, microfilaments connected to cellular m e m b r a n e , huge variety of m e m b r a n e proteins, higher content of polyunsaturated fatty acids (Bolis and Fange 1979), inability to transport D-glucose (Bolis et al. 1971)). The mechanism in which -SH groups of protein take part seems to be in agreement with the report by Barnes and Frieden, who showed that -SH blockers inhibited Cu 2+ -induced hemolysis. In conclusion, it emerges from this study that the action of oxygen radical species in the damage of cells is probably less important and the major cause of cell injury m a y be the alterations in protein structure.
Human rbc Carp rbc
0
0.05
0. I
0.3
4.43 -+ 0.13 3.01 -+ 0.23
4.36 -+ 0.13 2.99 -+ 0.28
4.07 a -+ 0.22 3.25 -+ 0.21
4.35 -+ 0.17 2.96 -+ 0.20
aControl vs 0.1 mmol/L p < 0.025
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