BIORHEOLOGY. 29; 217-229.1992 0006-355X/92 $5.00 + .00 Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
EFFECTS OF SUPEROXIDE ANIONS ON RED CELL DEFORMABILITY AND MEMBRANE PROTEINS
Nobuhiro Uyesaka 1, Setsuo Hasegawa 1, Noriaki Ishioka2 , Reiko Ishioka 1, Hideo Shi0 3 and Alan N. Schechter4 IDepartment of Physiology, Nippon Medical School, Sendagi 1-1-5, Bunkyo-ku, Tokyo 113, Japan, 2Division of Biochemistry, Institute of Medical Science, The Jikei University School of Medicine, Tokyo, Japan, 3Department of Neurology, Faculty of Medicine, Kyoto University, Kyoto, Japan, 4Laboratory of Chemical Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA
(Accepted 27.2.1992 by Editor N. Ohshima)
ABSTRACT The effect of superoxide anions (02) on red blood cells (RBC) deformability and membrane proteins was investigated using hypoxanthine-xanthine oxidase system. Exposure of RBC to 02 caused a marked decrease in RBC deformability with a concomitant increase in cell volume and shape changes. The RBC exposed to 02 also displayed pronounced degradation of membrane proteins such as band 3 protein and spectrin; new bands of low molecular weight products appeared as the original membrane proteins tended to diminish, without the appearance of high molecular weight products. Since the membrane proteins are involved in processes regulating membrane properties such as permeability and viscoelasticity, the decreased deform ability induced by 02 may be attributable to changes in membrane proteins. Interestingly, resealed ghosts exposed to 02 did not show any significant change in membrane proteins, which suggests the existence of further generation of 02 and subsequent production of other active oxygen species mediated by 02-initiated autoxidation of hemoglobin in intact RBC. Furthermore, electrophoretic analysis suggested that active oxygens increased the endogenous proteolytic susceptibility of RBC. In conclusion, a close linkage was suggested between RBC deformability and the membrane proteins.
KEY WORDS: red blood cells (RBC), superoxide anion, deformability, membrane protein, band 3 protein 217
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INTRODUCTION Superoxide anions (Oz) are indispensable in the maintenance of physiologic reactions and biological functions in vivo, such as the electron transfer system (redox cycling) and phagocytosis (1,2), but their excess generation and the subsequent production of other active oxygen species exert a variety of cytotoxic effects (1,3-6). Red blood cells (RBC) have been used extensively to elucidate the mechanism of peroxidative membrane damage at the cellular and molecular levels (7-10). RBC contain hemoglobin (Hb), which is a source of active oxygen species; that is, autoxidation of oxyhemoglobin (oxy-Hb) to methemoglobin (met-Hb) results in the release of Oz and the subsequent production of other active oxygens such as hydroxyl radical and hydrogen peroxide (7), because Oz is readily convertible to other active oxygen species (5). In RBC, therefore, the excess generation of Oz is regarded as a potential cause of oxidative injury that alters physiologic properties of RBC and, ultimately, their life span (7). Several lines of evidence suggest that the oxidative damage to RBC induces a change in physiologic functions such as RBC deformability (11-14); several studies reported decreased deformability due to the polymerization (cross-linking) of membrane proteins caused by active oxygen species and/or peroxide-generating compounds (15-17). To clarify further the mechanism of oxidative stress on RBC, we have studied the effect of Oz on RBC deformability and membrane proteins using the hypoxanthine-xanthine oxidase system. MATERIALS AND METHODS Materials
Xanthine oxidase was purchased from Boehringer Mannheim GmbH (Mannheim, Germany). Other reagents used were of reagent grade. All materials used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were from Pharmacia LKB (Bromma, Sweden). Preparation of RBC and resealed RBC ghosts
Venous blood from the antecubital vein of healthy young male adults was collected into a disposable syringe with an 18 gauge needle using 1/10 volume of 3.3% trisodium citrate as an anticoagulant. After centrifugation at 1300xg for 10 minutes, the plasma and buffy coat were carefully removed, and replaced with HEPES-buffered NaCl solution (HBS; 141 mM NaCl, 10 mM HEPES-Na buffer, 287 mOsm/kgH20, pH 7.4). RBC were washed three times by repeating the resuspension with HBS and the centrifugation at 800xg, 600xg, and 500xg, respectively.
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RBC ghosts were prepared from washed RBC, which were hemolyzed in 35 volumes of lysing buffered solution (LBS; 6 mM HEPES-Na, 1 mM MgCh, pH8.0) at O°C. The lysed cells were centrifuged at 44,400xg for 30 minutes at 2°C and the resulting pellets (unsealed RBC ghosts) were washed twice with LBS. Resealed RBC ghosts were prepared as follows. The unsealed ghost pellets (0.5 ml) were suspended in 35 ml of HBS, and were incubated at 36°C for 15 minutes. The resealed ghosts were collected by centrifugation at 17,800xg for 15 minutes at 22°C. Exposure of RBC and RBC ghosts to Oi
The intact RBC suspension was prepared with HBS, the hematocrit (Hct) value of which was adjusted to an adequate value for experiments. Similarly, the equivalent cell number of the RBC ghost suspension to the intact RBC suspension was prepared with HBS using an electronic hemocytometer (Nihon Koden, Celltac: MEK-4400, Tokyo, Japan). 30 mM of hypoxanthine solution (pH 7.4) was added to the sample suspension to a final concentration of 1.5 mM. After addition of xanthine oxidase, the mixture was incubated at 36°C for 1 hour in a waterbath with gentle shaking. The sample was cooled in ice before use for experiments. The generation of met-Hb resulting from Oi-triggered autoxidation of oxy-Hb was measured with a spectrophotometer (Hitachi, Model 356, Tokyo, Japan). RBC deformability
RBC deformability was investigated in terms of RBC filterability through the nickel mesh and viscosity of the RBC suspension. The Hct value of the RBC suspension was 10%. The filtration through the nickel mesh was performed by means of the vertical-tube method (18) with a minor modification (19). The filterability of the RBC was evaluated from pressure(P)-flow rate(Q) relationship of the RBC suspension constructed by the modified vertical-tube method (20). The nickel mesh (Dainippon Printing, Tokyo, Japan) is a new porous thin metal film produced to our specification using a photofabrication technique. We specified that the nickel mesh should have a diameter of 13 mm, a thickness of 11 !lm, a pore diameter of 5 !lm, an interpore distance of 35 !lm, and an effective filtration area of 0.50 cm 2 (21). Prior to the nickel mesh filtration experiment, the suspending medium was filtered through a Millipore filter with a pore size of 0.22 !lm to avoid otherwise inevitable microdust contamination. Since one set of the nickel mesh was reused for each experiment after an ultrasonic washing, all experimental data were obtained employing the same mesh. The viscosity of the RBC suspension was measured at 22°C by a cone-plate viscometer (Tokyokeiki, type E, Tokyo, Japan). Filtration experiments were carried out at room temperature (22±1.5°C) except for the preparation of samples.
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RBC shape and volume
An aliquot of the RBC suspension used in the experiments was fixed with isotonic 1% glutaraldehyde solution containing 50 mM of phosphate buffer (pH 7.4) and an adequate amount of NaCI at 4°C for 24 hours. The cells were dehydrated in a graded series of ethanol washes, dried at critical point, and coated with platinum/palladium. The specimens were observed in a scanning electron microscope (Hitachi, S-800, Tokyo, Japan) at IS kY. The change in cell volume was estimated from the measurement of packed cell volume (Hct value). The Hct value of the RBC suspension was 30% under the physiologic condition. Electrophoresis
SDS-PAGE of the ghost membrane proteins was carried out by the method of Laemmli (22). Exposure to Oi was carried out using the RBC suspension of 18% Hct value. The ghost membranes were solubilized to a final concentration of 2 mg(protein)/ml in SDS buffered solution (2% sodium dodecyl sulfate, 20% SUCrose, 20 mM Tris-HCI, pH 6.5) with 2% ~-mercaptoethanol. The solubilized membrane proteins (-60 f..lg) were loaded onto 10% polyacrylamide gels. The protein content of ghost sample was measured by the method of Lowry et al. (23). Electrophoresis was carried out for 3 hours at lo°C, using the LKB 2001 vertical electrophoresis unit (Pharmacia LKB, Bromma, Sweden). Gels were stained by 0.025% Coomassie Brilliant Blue R-250. The molecular weight of membrane proteins in the gels was estimated from the calibration curve of standard proteins, ranging from 18.5 kDa to 330 kDa. RESULTS Effect of Oi on RBC deformability
Figure lA shows P-Q relationship of Oi-treated RBC suspension through the nickel mesh. Based on Figure lA, Figure IB presents the flow rate at IS cmH20 plotted against the selected concentration of xanthine oxidase. The flow rate decreased as the concentration of xanthine oxidase increased. This indicates a dose-dependent decrease in the filterability of RBC exposed to Oi. The kinetic analysis of Figure IB based on the Michaelis-Menten equation showed that the Michaelis constant (Km) is 0.50 V/ml (xanthine oxidase). Exposure of RBC to Oi increased the cell volume; 7% increase in packed cell volume was observed at the concentration of 0.40 V/ml xanthine oxidase. RBC exposed to Oi also displayed dose-dependent shape changes; they became cup-shaped (stomatocytic) and triconcave-shaped (knizocytic) and then spherical with increasing concentration of xanthine oxidase (not shown). Oi-treated RBC exhibited a dark brownish color in a dose-dependent manner. Simple spectrophotometric analysis revealed the
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formation of met-Hb resulting from autoxidation of oxy-Hb, but no generation of hemichromes within the experimental sensitivity. The measurement of met-Hb using cyanide showed that met-Hb content was 35% at the concentration of 0.12 DIml xanthine oxidase.
A
B
Flow Rate ( ml/min) 25
Flow Rate at 15cmH,O (ml/min) 15 1/60 0.8
2 3
4 5
0.4
0
10
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\,
6 7
5 5
o
/
,./
/
.
.
/
to
-2
10 20 1/(xoxd)
~
o~o
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10 15 20 Pressure (cmH,O)
25
o
0.2
0.4 0.6 Xoxd (U/ml)
0.8
FIG. 1 Effects of super oxide anions on RBC filterability. A. Pressure-flow rate (P- Q ) relationship of RBC suspension of the selected concentration of xanthine oxidase(Xoxd) in the filtration through the nickel mesh. The hematocrit value of RBC suspension is 10%. 1: NaCl solution, 2: control, 3: 0.04 D/ml Xoxd, 4: 0.12 D/ml Xoxd, 5: 0.24 D/ml Xoxd, 6: 0040 D/ml Xoxd, 7: 0.80 D/ml Xoxd. B. Relationship between flow rate at 15 cmH20 and the concentration of xanthine oxidase. The plot was made based on the P-Q relationship shown in Fig. lA. The inserted figure shows Lineweaver-Burk plot for the decrement of flow rate (~Q) and the concentration of xanthine oxidase ([XoxdD obeying Michaelis-Menten kinetics. The Km value obtained from this plot was 0.50 Dim!.
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Figure 2A shows the viscosity of the Oi-treated RBC suspension. The viscosity of the RBC suspension increased as the concentration of xanthine oxidase increased, especially, at a low shear rate. Figure 2B presents both the viscosity at a shear rate of 19.2 sec- 1 and the square root of yield stress obtained from Casson's equation (24.a.b.) plotted against the selected concentration of xanthine oxidase. RBC exposed to 02 showed a dose-dependent increase in viscosity and yield stress. Km values for both viscosity and square root of yield stress were 0.50 Vlml, which was the same as value for the filtration experiment described above. A
B Viscosity at 19.2 sec- 1
Viscosity (cp)
Yield Stress't2 1;. (dyne/cm2) 2
(cp)
12
0.8
6 0.6
4
0.4
2
0.2
*
/
o
50
100
150
Shear Rate (1 / sec)
200
o
*
~,----~,--~,~--~--~,
o
0.2
0.4
0.6
0.8
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Xoxd (U / ml )
FIG. 2 Effects of super oxide anions on viscosity of RBC suspension.
A. Relationship between shear rate and apparent viscosity of RBC suspension of the selected concentration of xanthine oxidase (Xoxd). The hematocrit value of RBC suspension is 10%. 0 - 0 : control , . - . :0.04 Vlml Xoxd, .A.-.A. :0.12 Vlml Xoxd, 'f'-'f' :0.24 Vlml Xoxd, . - . :0.40 Vlml Xoxd, :0.80 Vlml Xoxd. B. Relationship between the apparent viscosity at shear rate of 19.2 sec- 1 (-tr--tr) or the square root of Casson's yield stress (*-*) and the concentration of xanthine oxidase. The value of Casson's yield stress was estimated from Casson's plot using the data shown in Fig. 2A.
*-*
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DEFORMABILliY, O2AND RBC
Effect of Oi on membrane proteins As shown in Figure 3A, RBC exposed to 02 exhibited a dose-dependent decrease in the main bands of membrane proteins, that is, spectrin (band 1, 2), ankyrin (band 2.1), band 3, band 4.1, band 4.2, actin (band 5) and band 6 without the appearance of high molecular weight materials. Band 3, ankyrin and band 4.2 were rapidly degraded and almost completely disappeared at the concentration of 0.08 Dlml xanthine oxidase (lane e in Fig. 3A). On the other hand, spectrin, band 4. 1, actin and band 6 were degraded more gradually, and the degradation continued at the concentration of 0.40 Dlml xanthine oxidase (lane h in Fig. 3A). In accordance with the initial rapid degradation of band 3 protein, new dominant bands of lower molecular weight (termed Nl: 50 kDa, N2: 41 kDa, N3: 37 kDa, N4: 22 kDa) appeared on the gel. The molecular weights of these new bands were similar to those of typical segments of band 3 protein produced by proteolytic enzymes (25,26). Attempts to assign other fragments of membrane proteins were
A
B
2 1i 2:1-
3-
4.14.2-
56-
-N1 -N2 ....... _ _ -N3
-------abcdefg
-N4
h
FIG. 3
abc
d
Effects of superoxide anions on membrane proteins investigated with SDS polyacrylamide gel electrophoresis. A. Intact RBC. a, control without hypoxanthine; b, control with hypoxanthine; c, 0.02 Dlml Xoxd; d, 0.04 Dlml Xoxd; e, 0.08 Dlml Xoxd; f, 0.16 Dlml Xoxd; g, 0.28 Dlml Xoxd; h, 0.40 Dlml Xoxd. NI, N2, N3 and N4 present new dominant bands. B. Resealed ghosts. a, control without hypoxanthine; b, 0.02 D/ml Xoxd; c, 0.08 Dlml Xoxd; d, 0.28 Dlml Xoxd.
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inconclusive. In contrast, resealed ghosts exposed to Oi did not show any marked change in bands on SDS-PAGE compared with the control (Fig. 3B). Furthermore, we confirmed that the membrane proteins was not degraded during the preparation of ghosts and during the incubation of the ghosts with SDS, after exposure to Oi.
DISCUSSION The major determinants of RBC deformability are cell geometry (especially, the cellular surface area to volume ratio and cell shape), viscoelastic properties of the cell membrane and intracellular fluid viscosity (27-29). Macroscopically, the decreased deformability induced by Oi, therefore, can be ascribed to the increase in cell volume and stomatocytic and knizocytic shape changes. In addition, yield stress obtained from Casson's plot increased with the increase in the concentration of xanthine oxidase, thereby suggesting that Oi increase cell-cell interactions, which usually increase yield stress as well as viscosity (24.b). Interestingly, Michaelis-Menten constants (Km) obtained from viscosity and yield stress coincide with that obtained from the filterability. This displays a close relationship among those rheological indices from biochemical and biorheological point of view, although the experimental measurements are different and the meaning what they suggest is not the same. In other words, this filtration measurement is useful as well as viscometry in detecting RBC deformability. It is well known that membrane proteins play an important role in membrane properties of RBC and accordingly, the physiologic functions of RBC such as membrane permeability (30,31) and RBC deform ability (27-29). In the present study, electrophoretic analysis of RBC exposed to Oi revealed pronounced degradation of membrane proteins without the appearance of protein cross-linking such as observed in photodynamic action (15) or exposure to hydroperoxide (16). The band 3 protein showed a drastic change accompanied by the appearance of new bands Nt, N2, N3 and N4. Band 3 protein is an integral, asymmetrically disposed, transmembrane protein (32), and is believed to be a channel or carrier of anion and water across the membrane (30,31). The increase in cell volume induced by Oi may be attributed to a resulting increase in water transport across the membrane, probably due to a structural change and degradation in band 3 protein. The electrophoretic alteration of peripheral proteins, that is, spectrin, ankyrin, band 4.1, band 4.2, and actin was also clearly detected. The molecular site of stability of cytoskeletal framework seems to involve the interactions between peripheral proteins (28,33). It is, therefore, possible that the perturbation of protein interactions changes the membrane properties such as viscoelasticity and consequently, RBC shape and deformability (27-29). Thus, microscopically, the decreased deformability induced by Oi can be attributed to changes in the molecular architecture of the cytoskeleton. Interestingly, it has been reported that
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the alteration of the membrane proteins seems to be a more sensitive indicator of cell exposure to oxygen radicals than is lipid peroxidation (10). SDS-PAGE of the resealed ghost exposed to Oz did not show any change in the membrane proteins. The result suggests that the oxidative degradation of membrane proteins is caused by intracellular Oz, especially by excess generation of intracellular Oz mediated by Oi-triggered autoxidation of Hb, including subsequent production of other active oxygen species such as hydroxyl radical and hydrogen peroxide (7). Therefore, the oxidative injury of RBC may be initiated from those areas on the membrane proteins that are adjacent to membrane-bound Hb. Thus, the marked degradation of band 3 protein already at a relatively low concentration of 02 can be ascribed to the oxidation of Hb, because the high affinity binding site of Hb is considered to be localized on band 3 protein (34,35). Similarly, rapid degradation of ankyrin and band 4.2, which are believed to be attached to band 3 protein (36), can be attributed to the oxidation of Hb. The appearance of the new bands (NI-N4), accompanied with progressive diminution of band 3 protein, closely resembles the series of discrete segments produced by the proteolytic digestion of band 3 protein; as far as stoichiometric assignments are concerned, the new band NI (50 kDa) resembled the C-terminal 52 kDa segment (extracellular fragment + intramembranous fragment) of band 3 protein produced by trypsin digestion, the new band N2 (41 kDa) resembled the cytoplasmic segment (41-42 kDa) generated by trypsin or papain digestion, the new band N3 (37 kDa) resembled the chymotryptic extracellular segment (38 kDa), and the new band N4 (22 kDa) resembled the cytoplasmic N-terminal segment by trypsin digestion (22 kDa) or S-cyanylation (23 kDa) (25,26). Although the structure of the segments produced by the exposure to 02 was not investigated by amino acid sequence analysis, the apparent new fragments (NI-N4) are considered to be derived from band 3 protein because of their nearly stoichiometric yields. Accordingly, the possible mechanism by which active oxygen species causes the degradation of membrane proteins is that active oxygen increases the proteolytic susceptibility of RBC, namely, activation of endogenous proteases or inactivation of the endogenous protease inhibitors. Indeed, the active oxygen species has been reported to cause extensive protein degradation (10,37), and proteolytic enzymes exist in RBC such as Ca2 +-activated neutral protease (calpain) which can digest band 4.1, band 3, spectrin (38), and ankyrin (39). Our results are summarized in Figure 4, which schematically shows a possible process for Oi-induced degradation of membrane proteins and decreased deformability of RBC. The findings suggest that Oi-triggered sequential oxidative reactions accompanied with autoxidation of Hb playa key role in the oxidative injury of RBC, and this injury includes endogenous proteolytic damage to membrane proteins as well as direct oxidative damage to the RBC membrane. This report is the first documentation showing the impaired RBC deformability due to
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the degradation of membrane proteins, not to the protein cross-linking. In conclusion, a close relationship is suggested between the physiologic functions of RBC and the structure of their membrane proteins, including a possible role of Hb in oxidative damage.
. decreased . d Increased ~ RBC deformability Increase ~ cell volume permeability t shape change
&
change in viscoelasticity
t
degradation of cy toskeleton
\ \ \
o·2- \\ \
Hb0 2
1-
MetHb
FIG. 4 A possible mechanism for the decreased deformability of RBC induced by 02-initiated sequential oxidative process of membrane proteins. 02-: superoxide anion, H202: hydrogen peroxide, OH-: hydroxyl radical, 102: singlet oxygen.
REFERENCES 1. CHANCE, B., SIBS, H. and BOVERIS, A. Hydroperoxide metabolism in mammalian organs. Physiol. Rev., 59,527-605,1979. 2. BELLAVITE, P. The superoxide-forming enzymatic system of phagocytes.
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Free Radical Biology & Medicine, 4, 225-261,1988. 3. KAPPUS, H. and SIES, H. Toxic drug effects associated with oxygen metabolism: Redox cycling and lipid peroxidation. Experientia, 37, 12331241,1981 4. FRIDOVICH, I. Superoxide radical: An endogenous toxicant. Ann. Rev. Pharmacal. Toxicol., 23,239-257, 1983. 5. HALLIWELL, B. and GUTTERIDGE, J.M.C. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. 1., 219, 1-14, 1984. 6. McCORD, J.M. Oxygen-derived free radicals in postischemic tissue injury. N. Engl. J. Med., 312, 159-163, 1985. 7. CARRELL, R.W., WINTERBOURN, C.C. and RACHMILEWITZ, E.A. Activated oxygen and haemolysis. Br.1. Haematol., 30, 259-264, 1975. 8. LYNGI, R.E. and FRIDOVICH, I. Effects of superoxide on the erythrocyte membrane. J. Bioi. Chem., 253, 1838-1845, 1978. 9. KONG, S. and DAVISON, A.J. The relative effectiveness of ·OH, H202, 02, and reducing free radicals in causing damage to biomembranes. A study of radiation damage to erythrocyte ghosts using selective free radical scavengers. Biochim. Biophys. Acta, 640,313-325, 1981. 10. DAVIES, K.J. A. and GOLDBERG, A.L. Oxygen radicals stimulate intracellular proteolysis and lipid peroxidation by independent mechanisms in erythrocytes. 1. Bioi. Chem.,262, 8220-8226, 1987. 11. JAIN, S.K., MOHANDAS, N., CLARK, M.R. and SHOHET, S.B. The effect of malonyldialdehyde, a product of lipid peroxidation, on the deformability, dehydration and 51Cr-survival of erythrocytes. Br. J. Haematol., 53, 247-255, 1983. 12. SNYDER, L.M., FORTIER, N.L., TRAINOR, J., JACOBS, J., LEB, L., LUBIN, B., CHIU, D., SHOHET, S. and MOHANDAS, N. Effect of hydrogen peroxide exposure on normal human erythrocyte deformability, morphology, surface characteristics, and spectrin-hemoglobin cross-linking. J. Clin. Invest., 76, 1971-1977, 1985. 13. CHIU, D. and LUBIN, B. Oxidative hemoglobin denaturation and RBC destruction: The effect of heme on red cell membranes. Semin. Hematol., 26, 128-135, 1989. 14. HEBBEL, R.P., LEUNG, A. and MOHANDAS, N. Oxidation-induced changes in microrheologic properties of the red blood cell membrane. Blood, 76, 1015-1020, 1990. 15. DUBBELMAN, T.M.A.R., De BRUIJNE, A.W. and Van STEVENINCK, J. Photodynamic effects of protoporphyrin on red blood cell deformability. Biochem. Biophys. Res. Commun., 77, 811-817, 1977. 16. CORRY, W.D., MEISELMAN, H.J. and HOCHSTEIN, P. t-butyl hydroperoxide-induced changes in the physicochemical properties of human erythrocytes. Biochim. Biophys. Acta, 597,224-234, 1980. 17. PFAFFEROTT, C, MEISELMAN, H.J. and HOCHSTEIN, P. The effect of malonyldialdehyde on erythrocyte deformability. Blood, 59, 12-15, 1982. 18. NICHOL, J., GIRLING, F., JERRARD, W., CLAXTON, E.B. and BURTON,
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AC. Fundamental instability of the small blood vessels and critical closing pressures in vascular beds. Am. 1. Physioi., 164, 330-344, 1951. 19. UYESAKA, N. Pressure-flow relationship of erythrocyte suspension in perfusion of nuclepore membrane and red cell deformability. Jpn. J. Physioi., 38, 145-158, 1988. 20. ARAI, K., IINO, M., SHIO, H. and UYESAKA, N. Further investigations of red cell deformability with nickel mesh. Biorheology, 27, 47-65, 1990. 21. ANEGA WA, T., SHIO, H., YASUDA, Y., FUJIMOTO, N. and KAMEY AMA, M. Erythrocyte deformability as measured with a newly developed nickel mesh. Clin. Remorheol., 7,803-810,1987. 22. LAEMMLI, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685, 1970. 23. LOWLY, O.H., ROSEBUROUGH, N.J., FARR, A.L. and RANDALL, R.J. Protein measurement with phenol reagent. J. Bioi. Chem., 193, 265-275, 1951. 24. a. MERRILL, E.W. and PELLETIER, G.A Viscosity of human blood: transition from Newtonian to non-Newtonian. J. Appl. Physioi., 23, 178182, 1967. b. MERRILL, E.W. Rheology of blood. Physioi. Rev., 49, 863-888, 1969. 25. STECK, T.L., RAMOS, B. and STRAPAZON, E. Proteolytic dissection of band 3, the predominant transmembrane polypeptide of the human erythroycyte membrane. Biochemistry, 15,1154-1161, 1976. 26. STECK, T.L., KOZIARZ, J.1., SINGH, M.K., REDDY, G. and KOHLER, H. Preparation and analysis of seven major, topographically defined fragments of band 3, the predominant transmembrane polypeptide of human erythrocyte membranes. Biochemistry, 17, 1216-1222, 1978. 27. MOHANDAS, N., CHASIS, J.A and SHOHET, S.B. The influence of membrane skeleton on red cell deformability, membrane material properties, and shape. Semin. Hematol., 20, 225-242, 1983. 28. SHEETZ, M.P. Membrane skeletal dynamics: Role in modulation of red cell deformability, mobility of transmembrane proteins, and shape. Semin. Hematoi., 20, 175-188, 1983. 29. CHIEN, S. Red cell deformability and its relevance to blood flow. Ann. Rev. Physioi., 49, 177-192, 1987. 30. BENZ, R., TOSTESON, M.T., SCHUBERT, D. Formation and properties of tetramers of band 3 protein from human erythrocyte membranes in planar lipid bilayers. Biochim. Biophys. Acta, 775,347-355, 1984. 31. LUKACOVIC, M.F., VERKMAN, A.S., DIX, J.A. and SOLOMON, AK. Specific interaction of the water transport inhibitor, pCMBS, with band 3 in red blood cell membranes. Biochim. Biophys. Acta, 778, 253-259, 1984. 32. LOW, P.S. Structure and function of the cytoplasmic domain of band 3: center of erythrocyte membrane-peripheral protein interactions. Biochim. Biophys. Acta, 864, 145-167, 1986. 33. BENNETT, V. Spectrin-based membrane skeleton: a multipotential adaptor between plasma membrane and cytoplasm. Physiol. Rev., 70, 1029-1065, 1990.
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34. SHAKLAI, N., YGUERABIDE, 1. and RANNEY, H.M. Classification and localization of hemoglobin binding sites of the red blood cell membrane. Biochemistry, 16, 5593-5597, 1977. 35. WALDER, 1.A., CHATTERJEE, R., STECK, T.L., LOW, P.S., MUSSO, G.F., KAISER, E.T., ROGERS, P.H. and ARNONE, A. The interaction of hemoglobin with the cytoplasmic domain of band 3 of the human erythrocyte membrane. J. Bioi. Chern., 259, 10238-10246, 1984. 36. KORSGREN, C. and COHEN, C.M. Associations of human erythrocyte band 4.2. J. Bioi. Chern., 263, 10212-10218, 1988. 37. DAVIES, KJ.A. Protein damage and degradation by oxygen radicals. J. Bioi. Chern., 262,9895-9901, 1987. 38. CROALL, D.E., MORROW, 1.S. and DeMARTINO, G.N. Limited proteolysis of the erythrocyte membrane skeleton by calcium-dependent proteinases. Biochirn. Biophys. Acta, 882, 287-296, 1986. 39. HALL, T.G. and BENNETT, V. Regulatory domains of erythrocyte ankyrin. 1. Bioi. Chern., 262, 10537-10545, 1987.
EFFECTS OF SUPEROXIDE ANIONS ON RED CELL DEFORMABILITY AND MEMBRANE PROTEINS
Nobuhiro Uyesaka 1, Setsuo Hasegawa 1, Noriaki Ishioka2 , Reiko Ishioka 1, Hideo Shi0 3 and Alan N. Schechter4 IDepartment of Physiology, Nippon Medical School, Sendagi 1-1-5, Bunkyo-ku, Tokyo 113, Japan, 2Division of Biochemistry, Institute of Medical Science, The Jikei University School of Medicine, Tokyo, Japan, 3Department of Neurology, Faculty of Medicine, Kyoto University, Kyoto, Japan, 4Laboratory of Chemical Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA
(Accepted 27.2.1992 by Editor N. Ohshima)
ABSTRACT The effect of superoxide anions (02) on red blood cells (RBC) deformability and membrane proteins was investigated using hypoxanthine-xanthine oxidase system. Exposure of RBC to 02 caused a marked decrease in RBC deformability with a concomitant increase in cell volume and shape changes. The RBC exposed to 02 also displayed pronounced degradation of membrane proteins such as band 3 protein and spectrin; new bands of low molecular weight products appeared as the original membrane proteins tended to diminish, without the appearance of high molecular weight products. Since the membrane proteins are involved in processes regulating membrane properties such as permeability and viscoelasticity, the decreased deform ability induced by 02 may be attributable to changes in membrane proteins. Interestingly, resealed ghosts exposed to 02 did not show any significant change in membrane proteins, which suggests the existence of further generation of 02 and subsequent production of other active oxygen species mediated by 02-initiated autoxidation of hemoglobin in intact RBC. Furthermore, electrophoretic analysis suggested that active oxygens increased the endogenous proteolytic susceptibility of RBC. In conclusion, a close linkage was suggested between RBC deformability and the membrane proteins.
KEY WORDS: red blood cells (RBC), superoxide anion, deformability, membrane protein, band 3 protein 217
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INTRODUCTION Superoxide anions (Oz) are indispensable in the maintenance of physiologic reactions and biological functions in vivo, such as the electron transfer system (redox cycling) and phagocytosis (1,2), but their excess generation and the subsequent production of other active oxygen species exert a variety of cytotoxic effects (1,3-6). Red blood cells (RBC) have been used extensively to elucidate the mechanism of peroxidative membrane damage at the cellular and molecular levels (7-10). RBC contain hemoglobin (Hb), which is a source of active oxygen species; that is, autoxidation of oxyhemoglobin (oxy-Hb) to methemoglobin (met-Hb) results in the release of Oz and the subsequent production of other active oxygens such as hydroxyl radical and hydrogen peroxide (7), because Oz is readily convertible to other active oxygen species (5). In RBC, therefore, the excess generation of Oz is regarded as a potential cause of oxidative injury that alters physiologic properties of RBC and, ultimately, their life span (7). Several lines of evidence suggest that the oxidative damage to RBC induces a change in physiologic functions such as RBC deformability (11-14); several studies reported decreased deformability due to the polymerization (cross-linking) of membrane proteins caused by active oxygen species and/or peroxide-generating compounds (15-17). To clarify further the mechanism of oxidative stress on RBC, we have studied the effect of Oz on RBC deformability and membrane proteins using the hypoxanthine-xanthine oxidase system. MATERIALS AND METHODS Materials
Xanthine oxidase was purchased from Boehringer Mannheim GmbH (Mannheim, Germany). Other reagents used were of reagent grade. All materials used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were from Pharmacia LKB (Bromma, Sweden). Preparation of RBC and resealed RBC ghosts
Venous blood from the antecubital vein of healthy young male adults was collected into a disposable syringe with an 18 gauge needle using 1/10 volume of 3.3% trisodium citrate as an anticoagulant. After centrifugation at 1300xg for 10 minutes, the plasma and buffy coat were carefully removed, and replaced with HEPES-buffered NaCl solution (HBS; 141 mM NaCl, 10 mM HEPES-Na buffer, 287 mOsm/kgH20, pH 7.4). RBC were washed three times by repeating the resuspension with HBS and the centrifugation at 800xg, 600xg, and 500xg, respectively.
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RBC ghosts were prepared from washed RBC, which were hemolyzed in 35 volumes of lysing buffered solution (LBS; 6 mM HEPES-Na, 1 mM MgCh, pH8.0) at O°C. The lysed cells were centrifuged at 44,400xg for 30 minutes at 2°C and the resulting pellets (unsealed RBC ghosts) were washed twice with LBS. Resealed RBC ghosts were prepared as follows. The unsealed ghost pellets (0.5 ml) were suspended in 35 ml of HBS, and were incubated at 36°C for 15 minutes. The resealed ghosts were collected by centrifugation at 17,800xg for 15 minutes at 22°C. Exposure of RBC and RBC ghosts to Oi
The intact RBC suspension was prepared with HBS, the hematocrit (Hct) value of which was adjusted to an adequate value for experiments. Similarly, the equivalent cell number of the RBC ghost suspension to the intact RBC suspension was prepared with HBS using an electronic hemocytometer (Nihon Koden, Celltac: MEK-4400, Tokyo, Japan). 30 mM of hypoxanthine solution (pH 7.4) was added to the sample suspension to a final concentration of 1.5 mM. After addition of xanthine oxidase, the mixture was incubated at 36°C for 1 hour in a waterbath with gentle shaking. The sample was cooled in ice before use for experiments. The generation of met-Hb resulting from Oi-triggered autoxidation of oxy-Hb was measured with a spectrophotometer (Hitachi, Model 356, Tokyo, Japan). RBC deformability
RBC deformability was investigated in terms of RBC filterability through the nickel mesh and viscosity of the RBC suspension. The Hct value of the RBC suspension was 10%. The filtration through the nickel mesh was performed by means of the vertical-tube method (18) with a minor modification (19). The filterability of the RBC was evaluated from pressure(P)-flow rate(Q) relationship of the RBC suspension constructed by the modified vertical-tube method (20). The nickel mesh (Dainippon Printing, Tokyo, Japan) is a new porous thin metal film produced to our specification using a photofabrication technique. We specified that the nickel mesh should have a diameter of 13 mm, a thickness of 11 !lm, a pore diameter of 5 !lm, an interpore distance of 35 !lm, and an effective filtration area of 0.50 cm 2 (21). Prior to the nickel mesh filtration experiment, the suspending medium was filtered through a Millipore filter with a pore size of 0.22 !lm to avoid otherwise inevitable microdust contamination. Since one set of the nickel mesh was reused for each experiment after an ultrasonic washing, all experimental data were obtained employing the same mesh. The viscosity of the RBC suspension was measured at 22°C by a cone-plate viscometer (Tokyokeiki, type E, Tokyo, Japan). Filtration experiments were carried out at room temperature (22±1.5°C) except for the preparation of samples.
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RBC shape and volume
An aliquot of the RBC suspension used in the experiments was fixed with isotonic 1% glutaraldehyde solution containing 50 mM of phosphate buffer (pH 7.4) and an adequate amount of NaCI at 4°C for 24 hours. The cells were dehydrated in a graded series of ethanol washes, dried at critical point, and coated with platinum/palladium. The specimens were observed in a scanning electron microscope (Hitachi, S-800, Tokyo, Japan) at IS kY. The change in cell volume was estimated from the measurement of packed cell volume (Hct value). The Hct value of the RBC suspension was 30% under the physiologic condition. Electrophoresis
SDS-PAGE of the ghost membrane proteins was carried out by the method of Laemmli (22). Exposure to Oi was carried out using the RBC suspension of 18% Hct value. The ghost membranes were solubilized to a final concentration of 2 mg(protein)/ml in SDS buffered solution (2% sodium dodecyl sulfate, 20% SUCrose, 20 mM Tris-HCI, pH 6.5) with 2% ~-mercaptoethanol. The solubilized membrane proteins (-60 f..lg) were loaded onto 10% polyacrylamide gels. The protein content of ghost sample was measured by the method of Lowry et al. (23). Electrophoresis was carried out for 3 hours at lo°C, using the LKB 2001 vertical electrophoresis unit (Pharmacia LKB, Bromma, Sweden). Gels were stained by 0.025% Coomassie Brilliant Blue R-250. The molecular weight of membrane proteins in the gels was estimated from the calibration curve of standard proteins, ranging from 18.5 kDa to 330 kDa. RESULTS Effect of Oi on RBC deformability
Figure lA shows P-Q relationship of Oi-treated RBC suspension through the nickel mesh. Based on Figure lA, Figure IB presents the flow rate at IS cmH20 plotted against the selected concentration of xanthine oxidase. The flow rate decreased as the concentration of xanthine oxidase increased. This indicates a dose-dependent decrease in the filterability of RBC exposed to Oi. The kinetic analysis of Figure IB based on the Michaelis-Menten equation showed that the Michaelis constant (Km) is 0.50 V/ml (xanthine oxidase). Exposure of RBC to Oi increased the cell volume; 7% increase in packed cell volume was observed at the concentration of 0.40 V/ml xanthine oxidase. RBC exposed to Oi also displayed dose-dependent shape changes; they became cup-shaped (stomatocytic) and triconcave-shaped (knizocytic) and then spherical with increasing concentration of xanthine oxidase (not shown). Oi-treated RBC exhibited a dark brownish color in a dose-dependent manner. Simple spectrophotometric analysis revealed the
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formation of met-Hb resulting from autoxidation of oxy-Hb, but no generation of hemichromes within the experimental sensitivity. The measurement of met-Hb using cyanide showed that met-Hb content was 35% at the concentration of 0.12 DIml xanthine oxidase.
A
B
Flow Rate ( ml/min) 25
Flow Rate at 15cmH,O (ml/min) 15 1/60 0.8
2 3
4 5
0.4
0
10
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\,
6 7
5 5
o
/
,./
/
.
.
/
to
-2
10 20 1/(xoxd)
~
o~o
5
10 15 20 Pressure (cmH,O)
25
o
0.2
0.4 0.6 Xoxd (U/ml)
0.8
FIG. 1 Effects of super oxide anions on RBC filterability. A. Pressure-flow rate (P- Q ) relationship of RBC suspension of the selected concentration of xanthine oxidase(Xoxd) in the filtration through the nickel mesh. The hematocrit value of RBC suspension is 10%. 1: NaCl solution, 2: control, 3: 0.04 D/ml Xoxd, 4: 0.12 D/ml Xoxd, 5: 0.24 D/ml Xoxd, 6: 0040 D/ml Xoxd, 7: 0.80 D/ml Xoxd. B. Relationship between flow rate at 15 cmH20 and the concentration of xanthine oxidase. The plot was made based on the P-Q relationship shown in Fig. lA. The inserted figure shows Lineweaver-Burk plot for the decrement of flow rate (~Q) and the concentration of xanthine oxidase ([XoxdD obeying Michaelis-Menten kinetics. The Km value obtained from this plot was 0.50 Dim!.
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Figure 2A shows the viscosity of the Oi-treated RBC suspension. The viscosity of the RBC suspension increased as the concentration of xanthine oxidase increased, especially, at a low shear rate. Figure 2B presents both the viscosity at a shear rate of 19.2 sec- 1 and the square root of yield stress obtained from Casson's equation (24.a.b.) plotted against the selected concentration of xanthine oxidase. RBC exposed to 02 showed a dose-dependent increase in viscosity and yield stress. Km values for both viscosity and square root of yield stress were 0.50 Vlml, which was the same as value for the filtration experiment described above. A
B Viscosity at 19.2 sec- 1
Viscosity (cp)
Yield Stress't2 1;. (dyne/cm2) 2
(cp)
12
0.8
6 0.6
4
0.4
2
0.2
*
/
o
50
100
150
Shear Rate (1 / sec)
200
o
*
~,----~,--~,~--~--~,
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0.2
0.4
0.6
0.8
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FIG. 2 Effects of super oxide anions on viscosity of RBC suspension.
A. Relationship between shear rate and apparent viscosity of RBC suspension of the selected concentration of xanthine oxidase (Xoxd). The hematocrit value of RBC suspension is 10%. 0 - 0 : control , . - . :0.04 Vlml Xoxd, .A.-.A. :0.12 Vlml Xoxd, 'f'-'f' :0.24 Vlml Xoxd, . - . :0.40 Vlml Xoxd, :0.80 Vlml Xoxd. B. Relationship between the apparent viscosity at shear rate of 19.2 sec- 1 (-tr--tr) or the square root of Casson's yield stress (*-*) and the concentration of xanthine oxidase. The value of Casson's yield stress was estimated from Casson's plot using the data shown in Fig. 2A.
*-*
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DEFORMABILliY, O2AND RBC
Effect of Oi on membrane proteins As shown in Figure 3A, RBC exposed to 02 exhibited a dose-dependent decrease in the main bands of membrane proteins, that is, spectrin (band 1, 2), ankyrin (band 2.1), band 3, band 4.1, band 4.2, actin (band 5) and band 6 without the appearance of high molecular weight materials. Band 3, ankyrin and band 4.2 were rapidly degraded and almost completely disappeared at the concentration of 0.08 Dlml xanthine oxidase (lane e in Fig. 3A). On the other hand, spectrin, band 4. 1, actin and band 6 were degraded more gradually, and the degradation continued at the concentration of 0.40 Dlml xanthine oxidase (lane h in Fig. 3A). In accordance with the initial rapid degradation of band 3 protein, new dominant bands of lower molecular weight (termed Nl: 50 kDa, N2: 41 kDa, N3: 37 kDa, N4: 22 kDa) appeared on the gel. The molecular weights of these new bands were similar to those of typical segments of band 3 protein produced by proteolytic enzymes (25,26). Attempts to assign other fragments of membrane proteins were
A
B
2 1i 2:1-
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-N1 -N2 ....... _ _ -N3
-------abcdefg
-N4
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FIG. 3
abc
d
Effects of superoxide anions on membrane proteins investigated with SDS polyacrylamide gel electrophoresis. A. Intact RBC. a, control without hypoxanthine; b, control with hypoxanthine; c, 0.02 Dlml Xoxd; d, 0.04 Dlml Xoxd; e, 0.08 Dlml Xoxd; f, 0.16 Dlml Xoxd; g, 0.28 Dlml Xoxd; h, 0.40 Dlml Xoxd. NI, N2, N3 and N4 present new dominant bands. B. Resealed ghosts. a, control without hypoxanthine; b, 0.02 D/ml Xoxd; c, 0.08 Dlml Xoxd; d, 0.28 Dlml Xoxd.
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inconclusive. In contrast, resealed ghosts exposed to Oi did not show any marked change in bands on SDS-PAGE compared with the control (Fig. 3B). Furthermore, we confirmed that the membrane proteins was not degraded during the preparation of ghosts and during the incubation of the ghosts with SDS, after exposure to Oi.
DISCUSSION The major determinants of RBC deformability are cell geometry (especially, the cellular surface area to volume ratio and cell shape), viscoelastic properties of the cell membrane and intracellular fluid viscosity (27-29). Macroscopically, the decreased deformability induced by Oi, therefore, can be ascribed to the increase in cell volume and stomatocytic and knizocytic shape changes. In addition, yield stress obtained from Casson's plot increased with the increase in the concentration of xanthine oxidase, thereby suggesting that Oi increase cell-cell interactions, which usually increase yield stress as well as viscosity (24.b). Interestingly, Michaelis-Menten constants (Km) obtained from viscosity and yield stress coincide with that obtained from the filterability. This displays a close relationship among those rheological indices from biochemical and biorheological point of view, although the experimental measurements are different and the meaning what they suggest is not the same. In other words, this filtration measurement is useful as well as viscometry in detecting RBC deformability. It is well known that membrane proteins play an important role in membrane properties of RBC and accordingly, the physiologic functions of RBC such as membrane permeability (30,31) and RBC deform ability (27-29). In the present study, electrophoretic analysis of RBC exposed to Oi revealed pronounced degradation of membrane proteins without the appearance of protein cross-linking such as observed in photodynamic action (15) or exposure to hydroperoxide (16). The band 3 protein showed a drastic change accompanied by the appearance of new bands Nt, N2, N3 and N4. Band 3 protein is an integral, asymmetrically disposed, transmembrane protein (32), and is believed to be a channel or carrier of anion and water across the membrane (30,31). The increase in cell volume induced by Oi may be attributed to a resulting increase in water transport across the membrane, probably due to a structural change and degradation in band 3 protein. The electrophoretic alteration of peripheral proteins, that is, spectrin, ankyrin, band 4.1, band 4.2, and actin was also clearly detected. The molecular site of stability of cytoskeletal framework seems to involve the interactions between peripheral proteins (28,33). It is, therefore, possible that the perturbation of protein interactions changes the membrane properties such as viscoelasticity and consequently, RBC shape and deformability (27-29). Thus, microscopically, the decreased deformability induced by Oi can be attributed to changes in the molecular architecture of the cytoskeleton. Interestingly, it has been reported that
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the alteration of the membrane proteins seems to be a more sensitive indicator of cell exposure to oxygen radicals than is lipid peroxidation (10). SDS-PAGE of the resealed ghost exposed to Oz did not show any change in the membrane proteins. The result suggests that the oxidative degradation of membrane proteins is caused by intracellular Oz, especially by excess generation of intracellular Oz mediated by Oi-triggered autoxidation of Hb, including subsequent production of other active oxygen species such as hydroxyl radical and hydrogen peroxide (7). Therefore, the oxidative injury of RBC may be initiated from those areas on the membrane proteins that are adjacent to membrane-bound Hb. Thus, the marked degradation of band 3 protein already at a relatively low concentration of 02 can be ascribed to the oxidation of Hb, because the high affinity binding site of Hb is considered to be localized on band 3 protein (34,35). Similarly, rapid degradation of ankyrin and band 4.2, which are believed to be attached to band 3 protein (36), can be attributed to the oxidation of Hb. The appearance of the new bands (NI-N4), accompanied with progressive diminution of band 3 protein, closely resembles the series of discrete segments produced by the proteolytic digestion of band 3 protein; as far as stoichiometric assignments are concerned, the new band NI (50 kDa) resembled the C-terminal 52 kDa segment (extracellular fragment + intramembranous fragment) of band 3 protein produced by trypsin digestion, the new band N2 (41 kDa) resembled the cytoplasmic segment (41-42 kDa) generated by trypsin or papain digestion, the new band N3 (37 kDa) resembled the chymotryptic extracellular segment (38 kDa), and the new band N4 (22 kDa) resembled the cytoplasmic N-terminal segment by trypsin digestion (22 kDa) or S-cyanylation (23 kDa) (25,26). Although the structure of the segments produced by the exposure to 02 was not investigated by amino acid sequence analysis, the apparent new fragments (NI-N4) are considered to be derived from band 3 protein because of their nearly stoichiometric yields. Accordingly, the possible mechanism by which active oxygen species causes the degradation of membrane proteins is that active oxygen increases the proteolytic susceptibility of RBC, namely, activation of endogenous proteases or inactivation of the endogenous protease inhibitors. Indeed, the active oxygen species has been reported to cause extensive protein degradation (10,37), and proteolytic enzymes exist in RBC such as Ca2 +-activated neutral protease (calpain) which can digest band 4.1, band 3, spectrin (38), and ankyrin (39). Our results are summarized in Figure 4, which schematically shows a possible process for Oi-induced degradation of membrane proteins and decreased deformability of RBC. The findings suggest that Oi-triggered sequential oxidative reactions accompanied with autoxidation of Hb playa key role in the oxidative injury of RBC, and this injury includes endogenous proteolytic damage to membrane proteins as well as direct oxidative damage to the RBC membrane. This report is the first documentation showing the impaired RBC deformability due to
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the degradation of membrane proteins, not to the protein cross-linking. In conclusion, a close relationship is suggested between the physiologic functions of RBC and the structure of their membrane proteins, including a possible role of Hb in oxidative damage.
. decreased . d Increased ~ RBC deformability Increase ~ cell volume permeability t shape change
&
change in viscoelasticity
t
degradation of cy toskeleton
\ \ \
o·2- \\ \
Hb0 2
1-
MetHb
FIG. 4 A possible mechanism for the decreased deformability of RBC induced by 02-initiated sequential oxidative process of membrane proteins. 02-: superoxide anion, H202: hydrogen peroxide, OH-: hydroxyl radical, 102: singlet oxygen.
REFERENCES 1. CHANCE, B., SIBS, H. and BOVERIS, A. Hydroperoxide metabolism in mammalian organs. Physiol. Rev., 59,527-605,1979. 2. BELLAVITE, P. The superoxide-forming enzymatic system of phagocytes.
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Free Radical Biology & Medicine, 4, 225-261,1988. 3. KAPPUS, H. and SIES, H. Toxic drug effects associated with oxygen metabolism: Redox cycling and lipid peroxidation. Experientia, 37, 12331241,1981 4. FRIDOVICH, I. Superoxide radical: An endogenous toxicant. Ann. Rev. Pharmacal. Toxicol., 23,239-257, 1983. 5. HALLIWELL, B. and GUTTERIDGE, J.M.C. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. 1., 219, 1-14, 1984. 6. McCORD, J.M. Oxygen-derived free radicals in postischemic tissue injury. N. Engl. J. Med., 312, 159-163, 1985. 7. CARRELL, R.W., WINTERBOURN, C.C. and RACHMILEWITZ, E.A. Activated oxygen and haemolysis. Br.1. Haematol., 30, 259-264, 1975. 8. LYNGI, R.E. and FRIDOVICH, I. Effects of superoxide on the erythrocyte membrane. J. Bioi. Chem., 253, 1838-1845, 1978. 9. KONG, S. and DAVISON, A.J. The relative effectiveness of ·OH, H202, 02, and reducing free radicals in causing damage to biomembranes. A study of radiation damage to erythrocyte ghosts using selective free radical scavengers. Biochim. Biophys. Acta, 640,313-325, 1981. 10. DAVIES, K.J. A. and GOLDBERG, A.L. Oxygen radicals stimulate intracellular proteolysis and lipid peroxidation by independent mechanisms in erythrocytes. 1. Bioi. Chem.,262, 8220-8226, 1987. 11. JAIN, S.K., MOHANDAS, N., CLARK, M.R. and SHOHET, S.B. The effect of malonyldialdehyde, a product of lipid peroxidation, on the deformability, dehydration and 51Cr-survival of erythrocytes. Br. J. Haematol., 53, 247-255, 1983. 12. SNYDER, L.M., FORTIER, N.L., TRAINOR, J., JACOBS, J., LEB, L., LUBIN, B., CHIU, D., SHOHET, S. and MOHANDAS, N. Effect of hydrogen peroxide exposure on normal human erythrocyte deformability, morphology, surface characteristics, and spectrin-hemoglobin cross-linking. J. Clin. Invest., 76, 1971-1977, 1985. 13. CHIU, D. and LUBIN, B. Oxidative hemoglobin denaturation and RBC destruction: The effect of heme on red cell membranes. Semin. Hematol., 26, 128-135, 1989. 14. HEBBEL, R.P., LEUNG, A. and MOHANDAS, N. Oxidation-induced changes in microrheologic properties of the red blood cell membrane. Blood, 76, 1015-1020, 1990. 15. DUBBELMAN, T.M.A.R., De BRUIJNE, A.W. and Van STEVENINCK, J. Photodynamic effects of protoporphyrin on red blood cell deformability. Biochem. Biophys. Res. Commun., 77, 811-817, 1977. 16. CORRY, W.D., MEISELMAN, H.J. and HOCHSTEIN, P. t-butyl hydroperoxide-induced changes in the physicochemical properties of human erythrocytes. Biochim. Biophys. Acta, 597,224-234, 1980. 17. PFAFFEROTT, C, MEISELMAN, H.J. and HOCHSTEIN, P. The effect of malonyldialdehyde on erythrocyte deformability. Blood, 59, 12-15, 1982. 18. NICHOL, J., GIRLING, F., JERRARD, W., CLAXTON, E.B. and BURTON,
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AC. Fundamental instability of the small blood vessels and critical closing pressures in vascular beds. Am. 1. Physioi., 164, 330-344, 1951. 19. UYESAKA, N. Pressure-flow relationship of erythrocyte suspension in perfusion of nuclepore membrane and red cell deformability. Jpn. J. Physioi., 38, 145-158, 1988. 20. ARAI, K., IINO, M., SHIO, H. and UYESAKA, N. Further investigations of red cell deformability with nickel mesh. Biorheology, 27, 47-65, 1990. 21. ANEGA WA, T., SHIO, H., YASUDA, Y., FUJIMOTO, N. and KAMEY AMA, M. Erythrocyte deformability as measured with a newly developed nickel mesh. Clin. Remorheol., 7,803-810,1987. 22. LAEMMLI, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685, 1970. 23. LOWLY, O.H., ROSEBUROUGH, N.J., FARR, A.L. and RANDALL, R.J. Protein measurement with phenol reagent. J. Bioi. Chem., 193, 265-275, 1951. 24. a. MERRILL, E.W. and PELLETIER, G.A Viscosity of human blood: transition from Newtonian to non-Newtonian. J. Appl. Physioi., 23, 178182, 1967. b. MERRILL, E.W. Rheology of blood. Physioi. Rev., 49, 863-888, 1969. 25. STECK, T.L., RAMOS, B. and STRAPAZON, E. Proteolytic dissection of band 3, the predominant transmembrane polypeptide of the human erythroycyte membrane. Biochemistry, 15,1154-1161, 1976. 26. STECK, T.L., KOZIARZ, J.1., SINGH, M.K., REDDY, G. and KOHLER, H. Preparation and analysis of seven major, topographically defined fragments of band 3, the predominant transmembrane polypeptide of human erythrocyte membranes. Biochemistry, 17, 1216-1222, 1978. 27. MOHANDAS, N., CHASIS, J.A and SHOHET, S.B. The influence of membrane skeleton on red cell deformability, membrane material properties, and shape. Semin. Hematol., 20, 225-242, 1983. 28. SHEETZ, M.P. Membrane skeletal dynamics: Role in modulation of red cell deformability, mobility of transmembrane proteins, and shape. Semin. Hematoi., 20, 175-188, 1983. 29. CHIEN, S. Red cell deformability and its relevance to blood flow. Ann. Rev. Physioi., 49, 177-192, 1987. 30. BENZ, R., TOSTESON, M.T., SCHUBERT, D. Formation and properties of tetramers of band 3 protein from human erythrocyte membranes in planar lipid bilayers. Biochim. Biophys. Acta, 775,347-355, 1984. 31. LUKACOVIC, M.F., VERKMAN, A.S., DIX, J.A. and SOLOMON, AK. Specific interaction of the water transport inhibitor, pCMBS, with band 3 in red blood cell membranes. Biochim. Biophys. Acta, 778, 253-259, 1984. 32. LOW, P.S. Structure and function of the cytoplasmic domain of band 3: center of erythrocyte membrane-peripheral protein interactions. Biochim. Biophys. Acta, 864, 145-167, 1986. 33. BENNETT, V. Spectrin-based membrane skeleton: a multipotential adaptor between plasma membrane and cytoplasm. Physiol. Rev., 70, 1029-1065, 1990.
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34. SHAKLAI, N., YGUERABIDE, 1. and RANNEY, H.M. Classification and localization of hemoglobin binding sites of the red blood cell membrane. Biochemistry, 16, 5593-5597, 1977. 35. WALDER, 1.A., CHATTERJEE, R., STECK, T.L., LOW, P.S., MUSSO, G.F., KAISER, E.T., ROGERS, P.H. and ARNONE, A. The interaction of hemoglobin with the cytoplasmic domain of band 3 of the human erythrocyte membrane. J. Bioi. Chern., 259, 10238-10246, 1984. 36. KORSGREN, C. and COHEN, C.M. Associations of human erythrocyte band 4.2. J. Bioi. Chern., 263, 10212-10218, 1988. 37. DAVIES, KJ.A. Protein damage and degradation by oxygen radicals. J. Bioi. Chern., 262,9895-9901, 1987. 38. CROALL, D.E., MORROW, 1.S. and DeMARTINO, G.N. Limited proteolysis of the erythrocyte membrane skeleton by calcium-dependent proteinases. Biochirn. Biophys. Acta, 882, 287-296, 1986. 39. HALL, T.G. and BENNETT, V. Regulatory domains of erythrocyte ankyrin. 1. Bioi. Chern., 262, 10537-10545, 1987.
