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Effect of ozone on erythrocyte membrane proteins
Ohemosphere No. 7, PP 443 - 448. ©Pergamon Press Ltd. 1979. Printed in Great Britain.
0045-6535/79/0701-nd43~02.00/0,
EFFECT OF OZONEON ERYTHROCYTE MEMBRANE PROTEINS H. Vem~eij and J. van Steveninck Sylvius Laboratories, Laboratory for Medical Chemistry, Wassenaarseweg 72, 2300 RA Leiden, The Netherlands INTRODUCTION Membrane damage of human erythrocytes by ozone is demonstrated by an increased osmotic f r a g i l i t y , loss of a c t i v i t y of membrane-bound enzymes, l i p i d peroxidation and cross-linking of membrane proteins 1-4. From some of these investigations i t has been concluded that free radical formation is responsible for the deleterious effects of ozone. Cross-linking of membrane proteins is considered to arise by reaction of proteins with l i p i d peroxidation aldehyde products4 or by protein radicals reacting with each other5. Chan e t a ] . showed that semicarbazide inhibited proteins cross-linking4. They ascribed this effect to trapping of malonaldehyde by semicarbazide. Previous studies from our laboratory have shown, however, that protoporphyrin-induced photodynamic cross-linking of membrane proteins is also inhibited by semicarbazide (unpublished results), whereas l i p i d peroxidation products are not involved in this process6'7. This fact threw doubt upon the conclusions of Chan et al. and therefore i t was decided to investigate the possible role of l i p i d peroxidation products in ozone-induced membrane protein cross-linking. In these studies i t has been shown that treatment of human erythrocyte membranes with ozone results in cross-linking of membrane proteins to high molecular aggregates, followed by degradation of these aggregates to small peptides. Moreover i t is concluded that l i p i d peroxidation is not involved in the cross-linking reaction of membrane proteins during ozone exposure.
METHODS Heparinized human blood was washed three times in isotonic phosphate buffered saline pH 7.4. Hemoglobin-free ghosts were prepared by the gradual osmotic method of Weed et al 8 • Spectrin was extracted from ghosts according to Fairbanks et al 9 and further purified by precipitation at pH 5.1. The protein pellet was dissolved
443
444
No. 7
again to 0.075 mg/ml in 4.8 mMTris-HCl buffer (pH 7.4) containing 0.85 mM EDTA and 10.2 mM NaCl. Protein concentrations were determined by the method of Lowry et al I0 with bovine serum albumin as as standard. Ozone was generated in a i r in a Fischer Ozone Generator OZ I I I at a rate of 12 ~mol/min with an airflow of 17 ml/min. Ozone production was measured by t i t r a t i o n with KI-Na2S203 at pH 7.0. Treatment of ghosts and spectrin with ozone was carried out by bubbling the gasstream into 2 ml samples to which 4 ul Silicon-defoaming agent (Fluka A.G.) had been added. This antifoaming agent had no effects on any of the results. Polyacrylamide gel electrophoresis was performed as described by Fairbanks et al 9. Prior to electrophoresis or gel chromatography the protein samples were dissolved and incubated for 30 min at 370 C in 10 mMTris-HCl (pH 8.0) containing 1 mM EDTA, 40 mM d i t h i o t h r e i t o l and 1% SDS, to a protein content of about 0.6 mg/ml f o r electrophoresis and of 2 mg/ml for gel chromatography. Gel chromatography was carried out by applying 2 ml of the protein solution to a Sepharose 4B column (100 x 1.5 cm) and elution with 10 mMTris-HCl (pH 7.4), containing 1% SDS.
Lipid peroxidation by H202 was performed according to Stocks and Dormandy11 Malonaldehyde was prepared by acid hydrolysis of i , I, 3, 3-tetramethoxypropane as described by Girotti 12. Malonaldehyde concentrations were measured with thiobarbituric acid, as described by Ottolenghi 13.
RESULTS The effect of ozone on ghost proteins is shown in f i g . 1. I t can be seen that there appear high molecular aggregates (MW > 106) on top of the gels, with a concomitant loss of a l l protein bands. This pehnomenon of protein cross-linking by ozone is not restricted to ghost preparations. When intact red blood cells were exposed to ozone t i l l
about 50% of the i n t r a c e l l u l a r K+ had leaked out of the cells, with
subsequent ghost preparation and gel electrophoresis, similar protein cross-linking patterns were observed. This protein cross-linking is not caused by formation of disulfide bands, since reduction with 40 mM d i t h i o t r e i t o l was carried out prior to electrophoresis.
Lipid peroxidation took place during exposure of ghosts to ozone, as indicated by the generation of malonaldehyde in concentrations up to 0.03 mM. Lipid peroxidation by treatment of the membranes with H202 resulted in the generation of malonaldehyde in concentrations of 0.03 - 0.05 mM, but without any protein cross-linking.
No. 7
445
Fi~.l. Effect of ozone on erythrocyte membrane proteins (2.5 mg protein/ml) as revealed by SDS-polyacrylamide gel electrophoresis. Exposure to ozone during 0 (a), 1 (b), 2(c), 5 (d), 10 (e) and 15 (f) rain.
Fi~.2. Malonaldehyde induced cross-linking of spectrin after 5 min incubation at pH 7.4. Malonaldehyde concentration: a, control (0 mM); b, 0.15 mM; c, 1.5 mM; d, 15 mM.
Malonaldehyde added to ghost suspensions or spectrin solutions caused a slowly progressive protein cross-linking i f added at concentrations of 1 mM or higher (fig. 2). Solubilized spectrin was strongly cross-linked by exposure to ozone (fig. 3A), without detectable formation of malonaldehyde. L •
Fig. 3. Protection against ozone-induced cross-linking of eluted spectrin by semicarbazide. Ozone exposure times: 0 (a), 0.5 (b), 1 (c), 2 (d) and 5 (e) min.
!iiiijiiiiii !ii! ii! i!il
A. control experiment
i¸¢iii!
B. in the prasence of 40 mM semi carbazide.
iii iil/i!iilCiiii i!!i!!ii~ii!]i¢iI!~i~i%i!ii iiii!! iii!i!!ii ~j~ ~ij~i • ~Li~ ~!!~i~k
i!i! ~
ii~ iiii: ~ii ~i~%!!iiiii~il ¸iii~!!i~ii :iliil ii: iii~~
~ ~ ii~i¸ili
!~iiii!i!!iil
iiiii!iiiii!
446
No. 7
In further experiments the strong protection by semicarbazide against ozone-induced
protein cross-linking could be confirmed. This protection was not only found in ghost suspensions but also with isolated spectrin (fig. 3B). No protection could be observed, however, when the semicarbazide was exposed to ozone before adding i t to the gost or spectrin sample. In experiments with 14C-labelled semicarbazide i t could be shown that this agent reacts with ozone, giving at least two reaction products (fig. 4).
--"
sotvent
front
"--
0.6
A2m 0.,;
4
i O.Z.
!
i?,
i
,
~0 or~n
..........................
Autoradiography of a silicagel yer chromatogram of 14C-semicarbazide exposed to ozone in 10 m M p h o s phate buffer pH 7.4. Semicarbazide concentration: I mM. Exposure times: 0 - 5 - 10 - 20 min. Solvent system: n-butanol-acetic acid-water (4 : 1 : 1 v/v/v).
'%o
5o ml ol effluenl
Ibo
Fi .5. Chromatography of a spectrin solution ~ ( ~ ) and after exposure to ozone @ - o - - - ~ on a sepharose 4B column. Arrows indicate void volume and total volume.
After prolonged exposure of ghosts to ozone a loss of all protein bands, including the cross-linked complex, from the electropherogram was obvious, although no decrease in protein content was measured, using the method of Lowry et al. Gel f i l t r a tion of the solubilized protein samples on Sepharose-4B showed that subsequent to ozone exposure there appeared a pool of polypeptides with molecular weights between 104 - 2.104 (fig. 5). On electropherograms these peptides could only be visualized when fixation was carried out in 30% instead of 10% trichloroacetic acid.
No. ?
44?
DISCUSSION The deleterious effects of ozone on red blood cell membranes are reflected by l i p i d peroxidation, inactivation of membrane-bound enzymes and membrane protein crosslinking. Chan et al. ascribed the membrane protein cross-linking to a reaction of the proteins with malonaldehyde, produced as a consequence of l i p i d peroxidation. They based this conclusion on the known protein cross-linking properties of aldehydes and the protection by semicarbazide, supposed to be based on trapping of aldehydes by this
agent 4.
The protection against cross-linking by semicarbazide is, however, no conclusive argument in favour of this mechanism. Protoporphyrin-induced photodynamic crosslinking of membrane proteins is also inhibited by semicarbazide, whereas l i p i d peroxidation is not involved in this process6'7. Moreover, as shown in this paper, semicarbazide also inhibits cross-linking of solubilized spectrin, whereas this solution contains no, or only trace amounts of lipids 14. Accordingly no malonaldehyde generation was found in ozone-exposed spectrin samples. These results show that in these experiments the inhibition of cross-linking by semicarbazide cannot be explained by malonaldehyde trapping. More l i k e l y the protection is based on the direct reaction between this reagent and ozone, as shown in f i g . 4. The other experimental results also contradict an essential role of l i p i d peroxidation in protein cross-linking. H2O2-induced malonaldehyde generation in the membrane structure to the same or even higher levels than observed during ozone exposure did not evoke membrane protein cross-linking. Finally, malonaldehyde added to the medium only induced some protein cross-linking, i f added at concentrations much higher than those observed during ozone exposure. These results indicate that membrane protein cross-linking is not the consequence of l i p i d peroxidation, but is caused by a direct effect of ozone on the proteins. After prolonged exposure to ozone a degradation to small peptides was observed ( f i g . 5). Presumably this reflects oxidative cleavage of peptide bands. The chemical nature of both the cross-linking and the degradation reactions w i l l be the subject of further studies.
ACKNOWLEDGEMENTS The authors are much indepted to Miss Hennie Vreeburg for her technical assistance.
This work was supported by the Netherlands Foundation for Fundamental
Medical Research (FUNGO, grant 13-39-28).
448
No. 7
REFERENCES 1.
R. Brinkman, H.B. Lamberts and T.S. Veninga, Lancet, 1 (1964) 133-136.
2.
B.D. Goldstein and O.J. Balchum, Proc. Soc. Exp. Biol. Med., 126 (1967) 356-358.
3.
R.D. Buckley, J.C. Hackney, K. Clark and C. Posin, Arch. Environm. Health,
4.
30 ~1975) 40-43. P.C. Chan, R.J. Kindya and L. Kesner, J. Biol. Chem., 252 (23) (1977) 8537-8541.
5.
H. Nielsen, Lipids, 13 (4) (1978) 253-258.
6.
A.F.P.M. de Goeij, R.J.C. van Straalen and J. van Steveninck, Clin. Chim. Acta, 71 (1976) 485-494.
7.
T.M.A.R. Dubbelman, A.F.P.M. de Goeij and J. van Steveninck, Biochim. Biophys. Acta, 511 (1978) 141-151.
8.
R.I. Weed, C.F. Reed and G. Berg, J. Clin. Invest., 42 (1963) 581-588.
9.
G. Fairbanks, T.L. Steck and D.F.H. Wallach, Biochemistry, 10 (1971) 2606-2617.
10. O.H. Lowry, N.J. Rosenbrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265-275. 11. J. Stocks and T.L. Domandy, B r i t . J. Haematol., 20 (1971) 95-111. 12. A.W. G i r o t t i , Biochemistry, 14 (1975) 3377-3383. 13. A. Ottolenghi, Arch. Biochem. Biophys., 79 (1959) 355-363. 14. F. Weidehamm, B. Brdiczka and M. Wildermuth, Mol. Biol. Rep., 4 (1978) 25-28.
(Received in The Netherlands 24 May 1979)
0045-6535/79/0701-nd43~02.00/0,
EFFECT OF OZONEON ERYTHROCYTE MEMBRANE PROTEINS H. Vem~eij and J. van Steveninck Sylvius Laboratories, Laboratory for Medical Chemistry, Wassenaarseweg 72, 2300 RA Leiden, The Netherlands INTRODUCTION Membrane damage of human erythrocytes by ozone is demonstrated by an increased osmotic f r a g i l i t y , loss of a c t i v i t y of membrane-bound enzymes, l i p i d peroxidation and cross-linking of membrane proteins 1-4. From some of these investigations i t has been concluded that free radical formation is responsible for the deleterious effects of ozone. Cross-linking of membrane proteins is considered to arise by reaction of proteins with l i p i d peroxidation aldehyde products4 or by protein radicals reacting with each other5. Chan e t a ] . showed that semicarbazide inhibited proteins cross-linking4. They ascribed this effect to trapping of malonaldehyde by semicarbazide. Previous studies from our laboratory have shown, however, that protoporphyrin-induced photodynamic cross-linking of membrane proteins is also inhibited by semicarbazide (unpublished results), whereas l i p i d peroxidation products are not involved in this process6'7. This fact threw doubt upon the conclusions of Chan et al. and therefore i t was decided to investigate the possible role of l i p i d peroxidation products in ozone-induced membrane protein cross-linking. In these studies i t has been shown that treatment of human erythrocyte membranes with ozone results in cross-linking of membrane proteins to high molecular aggregates, followed by degradation of these aggregates to small peptides. Moreover i t is concluded that l i p i d peroxidation is not involved in the cross-linking reaction of membrane proteins during ozone exposure.
METHODS Heparinized human blood was washed three times in isotonic phosphate buffered saline pH 7.4. Hemoglobin-free ghosts were prepared by the gradual osmotic method of Weed et al 8 • Spectrin was extracted from ghosts according to Fairbanks et al 9 and further purified by precipitation at pH 5.1. The protein pellet was dissolved
443
444
No. 7
again to 0.075 mg/ml in 4.8 mMTris-HCl buffer (pH 7.4) containing 0.85 mM EDTA and 10.2 mM NaCl. Protein concentrations were determined by the method of Lowry et al I0 with bovine serum albumin as as standard. Ozone was generated in a i r in a Fischer Ozone Generator OZ I I I at a rate of 12 ~mol/min with an airflow of 17 ml/min. Ozone production was measured by t i t r a t i o n with KI-Na2S203 at pH 7.0. Treatment of ghosts and spectrin with ozone was carried out by bubbling the gasstream into 2 ml samples to which 4 ul Silicon-defoaming agent (Fluka A.G.) had been added. This antifoaming agent had no effects on any of the results. Polyacrylamide gel electrophoresis was performed as described by Fairbanks et al 9. Prior to electrophoresis or gel chromatography the protein samples were dissolved and incubated for 30 min at 370 C in 10 mMTris-HCl (pH 8.0) containing 1 mM EDTA, 40 mM d i t h i o t h r e i t o l and 1% SDS, to a protein content of about 0.6 mg/ml f o r electrophoresis and of 2 mg/ml for gel chromatography. Gel chromatography was carried out by applying 2 ml of the protein solution to a Sepharose 4B column (100 x 1.5 cm) and elution with 10 mMTris-HCl (pH 7.4), containing 1% SDS.
Lipid peroxidation by H202 was performed according to Stocks and Dormandy11 Malonaldehyde was prepared by acid hydrolysis of i , I, 3, 3-tetramethoxypropane as described by Girotti 12. Malonaldehyde concentrations were measured with thiobarbituric acid, as described by Ottolenghi 13.
RESULTS The effect of ozone on ghost proteins is shown in f i g . 1. I t can be seen that there appear high molecular aggregates (MW > 106) on top of the gels, with a concomitant loss of a l l protein bands. This pehnomenon of protein cross-linking by ozone is not restricted to ghost preparations. When intact red blood cells were exposed to ozone t i l l
about 50% of the i n t r a c e l l u l a r K+ had leaked out of the cells, with
subsequent ghost preparation and gel electrophoresis, similar protein cross-linking patterns were observed. This protein cross-linking is not caused by formation of disulfide bands, since reduction with 40 mM d i t h i o t r e i t o l was carried out prior to electrophoresis.
Lipid peroxidation took place during exposure of ghosts to ozone, as indicated by the generation of malonaldehyde in concentrations up to 0.03 mM. Lipid peroxidation by treatment of the membranes with H202 resulted in the generation of malonaldehyde in concentrations of 0.03 - 0.05 mM, but without any protein cross-linking.
No. 7
445
Fi~.l. Effect of ozone on erythrocyte membrane proteins (2.5 mg protein/ml) as revealed by SDS-polyacrylamide gel electrophoresis. Exposure to ozone during 0 (a), 1 (b), 2(c), 5 (d), 10 (e) and 15 (f) rain.
Fi~.2. Malonaldehyde induced cross-linking of spectrin after 5 min incubation at pH 7.4. Malonaldehyde concentration: a, control (0 mM); b, 0.15 mM; c, 1.5 mM; d, 15 mM.
Malonaldehyde added to ghost suspensions or spectrin solutions caused a slowly progressive protein cross-linking i f added at concentrations of 1 mM or higher (fig. 2). Solubilized spectrin was strongly cross-linked by exposure to ozone (fig. 3A), without detectable formation of malonaldehyde. L •
Fig. 3. Protection against ozone-induced cross-linking of eluted spectrin by semicarbazide. Ozone exposure times: 0 (a), 0.5 (b), 1 (c), 2 (d) and 5 (e) min.
!iiiijiiiiii !ii! ii! i!il
A. control experiment
i¸¢iii!
B. in the prasence of 40 mM semi carbazide.
iii iil/i!iilCiiii i!!i!!ii~ii!]i¢iI!~i~i%i!ii iiii!! iii!i!!ii ~j~ ~ij~i • ~Li~ ~!!~i~k
i!i! ~
ii~ iiii: ~ii ~i~%!!iiiii~il ¸iii~!!i~ii :iliil ii: iii~~
~ ~ ii~i¸ili
!~iiii!i!!iil
iiiii!iiiii!
446
No. 7
In further experiments the strong protection by semicarbazide against ozone-induced
protein cross-linking could be confirmed. This protection was not only found in ghost suspensions but also with isolated spectrin (fig. 3B). No protection could be observed, however, when the semicarbazide was exposed to ozone before adding i t to the gost or spectrin sample. In experiments with 14C-labelled semicarbazide i t could be shown that this agent reacts with ozone, giving at least two reaction products (fig. 4).
--"
sotvent
front
"--
0.6
A2m 0.,;
4
i O.Z.
!
i?,
i
,
~0 or~n
..........................
Autoradiography of a silicagel yer chromatogram of 14C-semicarbazide exposed to ozone in 10 m M p h o s phate buffer pH 7.4. Semicarbazide concentration: I mM. Exposure times: 0 - 5 - 10 - 20 min. Solvent system: n-butanol-acetic acid-water (4 : 1 : 1 v/v/v).
'%o
5o ml ol effluenl
Ibo
Fi .5. Chromatography of a spectrin solution ~ ( ~ ) and after exposure to ozone @ - o - - - ~ on a sepharose 4B column. Arrows indicate void volume and total volume.
After prolonged exposure of ghosts to ozone a loss of all protein bands, including the cross-linked complex, from the electropherogram was obvious, although no decrease in protein content was measured, using the method of Lowry et al. Gel f i l t r a tion of the solubilized protein samples on Sepharose-4B showed that subsequent to ozone exposure there appeared a pool of polypeptides with molecular weights between 104 - 2.104 (fig. 5). On electropherograms these peptides could only be visualized when fixation was carried out in 30% instead of 10% trichloroacetic acid.
No. ?
44?
DISCUSSION The deleterious effects of ozone on red blood cell membranes are reflected by l i p i d peroxidation, inactivation of membrane-bound enzymes and membrane protein crosslinking. Chan et al. ascribed the membrane protein cross-linking to a reaction of the proteins with malonaldehyde, produced as a consequence of l i p i d peroxidation. They based this conclusion on the known protein cross-linking properties of aldehydes and the protection by semicarbazide, supposed to be based on trapping of aldehydes by this
agent 4.
The protection against cross-linking by semicarbazide is, however, no conclusive argument in favour of this mechanism. Protoporphyrin-induced photodynamic crosslinking of membrane proteins is also inhibited by semicarbazide, whereas l i p i d peroxidation is not involved in this process6'7. Moreover, as shown in this paper, semicarbazide also inhibits cross-linking of solubilized spectrin, whereas this solution contains no, or only trace amounts of lipids 14. Accordingly no malonaldehyde generation was found in ozone-exposed spectrin samples. These results show that in these experiments the inhibition of cross-linking by semicarbazide cannot be explained by malonaldehyde trapping. More l i k e l y the protection is based on the direct reaction between this reagent and ozone, as shown in f i g . 4. The other experimental results also contradict an essential role of l i p i d peroxidation in protein cross-linking. H2O2-induced malonaldehyde generation in the membrane structure to the same or even higher levels than observed during ozone exposure did not evoke membrane protein cross-linking. Finally, malonaldehyde added to the medium only induced some protein cross-linking, i f added at concentrations much higher than those observed during ozone exposure. These results indicate that membrane protein cross-linking is not the consequence of l i p i d peroxidation, but is caused by a direct effect of ozone on the proteins. After prolonged exposure to ozone a degradation to small peptides was observed ( f i g . 5). Presumably this reflects oxidative cleavage of peptide bands. The chemical nature of both the cross-linking and the degradation reactions w i l l be the subject of further studies.
ACKNOWLEDGEMENTS The authors are much indepted to Miss Hennie Vreeburg for her technical assistance.
This work was supported by the Netherlands Foundation for Fundamental
Medical Research (FUNGO, grant 13-39-28).
448
No. 7
REFERENCES 1.
R. Brinkman, H.B. Lamberts and T.S. Veninga, Lancet, 1 (1964) 133-136.
2.
B.D. Goldstein and O.J. Balchum, Proc. Soc. Exp. Biol. Med., 126 (1967) 356-358.
3.
R.D. Buckley, J.C. Hackney, K. Clark and C. Posin, Arch. Environm. Health,
4.
30 ~1975) 40-43. P.C. Chan, R.J. Kindya and L. Kesner, J. Biol. Chem., 252 (23) (1977) 8537-8541.
5.
H. Nielsen, Lipids, 13 (4) (1978) 253-258.
6.
A.F.P.M. de Goeij, R.J.C. van Straalen and J. van Steveninck, Clin. Chim. Acta, 71 (1976) 485-494.
7.
T.M.A.R. Dubbelman, A.F.P.M. de Goeij and J. van Steveninck, Biochim. Biophys. Acta, 511 (1978) 141-151.
8.
R.I. Weed, C.F. Reed and G. Berg, J. Clin. Invest., 42 (1963) 581-588.
9.
G. Fairbanks, T.L. Steck and D.F.H. Wallach, Biochemistry, 10 (1971) 2606-2617.
10. O.H. Lowry, N.J. Rosenbrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265-275. 11. J. Stocks and T.L. Domandy, B r i t . J. Haematol., 20 (1971) 95-111. 12. A.W. G i r o t t i , Biochemistry, 14 (1975) 3377-3383. 13. A. Ottolenghi, Arch. Biochem. Biophys., 79 (1959) 355-363. 14. F. Weidehamm, B. Brdiczka and M. Wildermuth, Mol. Biol. Rep., 4 (1978) 25-28.
(Received in The Netherlands 24 May 1979)