Download Increased Susceptibility of the Sickle Cell Membrane

From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
+
Increased Susceptibility of the Sickle Cell Membrane Ca2+ Mg2+-ATPaseto
t-Butylhydroperoxide: Protective Effects of Ascorbate and Desferal
By R. Blaine Moore, Todd M. Hulgan, Jerril W. Green, and Lucy D. Jenkins
Normal and sickle cell erythrocyte membranes were examined for significant differences in their ATPase activities,
thiobarbituric acid reactive products formed (measured relative t o malondialdehyde), membrane protein polymerization,
and number of protein-free sulfhydryl groups when treated
with 0.5 mmol/L t-butylhydroperoxide (tBHP)for 30 minutes.
Isolated sickle cell membranes treated with tBHP produced
significantly greater inhibition in both their basal and calmodulin-stimulated Caz+ Mg2+-ATPaseactivities (75% inhibition in both cases) compared with that of control membranes. I n addition, there was significantly more
malondialdehyde formed from sickle cell membranes compared with control membranes. Oxidation caused greater
protein polymerization in sickle cell membranes compared
with normal membranes as demonstrated by the formation
of high molecular weight polymers separated on sodium
dodecyl sulfate polyacrylamide gels. The number of free
sulfhydryl groups present in spectrin and actin decreased
more in sickle cell membranes as measured by ’H-N-ethyl
maleimide autoradiography and gel scanning. To prevent
enzyme inhibition, erythrocyte membranes were treated
with tBHP in the presenceof 1mmol/L ascorbate, a potential
antioxidant, and 1 mmol/L desferal, an iron chelator. Both
ascorbate and desferal added alone with tBHP were effective
in preventing inhibition of the basal and calmodulin-stimulated Ca2+ Mg2’-ATPase activities in normal membranes,
but in sickle cell membranes only the addition of ascorbate
and desferal together offered significant protection. The
enhanced oxidation observed with sickle cell membranes can
be mimicked in normal white membranes by adding hemoglobin, hemin, or ferrous chloride in the presence of tBHP. In
contrast t o hemoglobin, ferrous chloride has the ability t o
enhance membrane oxidation in the presence of ascorbate
with or without tBHP. Furthermore, the addition of desferal
t o these membranes greatly decreased the iron-ascorbatetBHP oxidation of erythrocyte membranes as determined by
the sustained ATPase activities and the reduced formation of
malondialdehyde. Maximal protection was provided by 1
mmol/L desferal in the presence of 1 mmol/L ascorbate,
although some protection was observed even at 10 pmol/L,
the lowest concentration tested. These results are discussed
in light of the pro- and anti-oxidant effects of ascorbate in the
absence and presence of iron and tBHP.
0 1992by The American Society of Hematology.
E
idants such as vitamin C, vitamin E, and riboflavin are
decreased in subjects with sickle cell anemia.6-9 Lipid
peroxidation” and reduction in the membrane free sulfhydryl status” have been reported as changes in sickle cells
resulting from the proposed oxidative stress.
Exposure of erythrocytes or erythrocyte membranes to
hydroperoxides, particularly t-butylhydroperoxide (tBHP),
can result in a number of membrane changes including lipid
peroxidation,I2malondialdehyde f~rmation,”.’~
protein polymeri~ation,’~.’~
and protein amino acid degradation.I6
Enzymes are also inhibited by reactive oxygen species and
the Ca2+-ATPaseof erythrocyte membranes is one of the
most sensitive of these enzymes.”
The Ca2+-ATPaseis an integral membrane protein with a
molecular weight of 140,000 daltons.” The enzyme is
inhibited by sulfhydryl alkylating agents” and by diamide, a
selective sulfhydryl oxidizing agent.” The presence of
phospholipids is required for optimal activity of the Ca2+but inhibition of the enzyme by hydroperoxides
can occur without significant lipid peroxidation.”.” Hydroperoxide inhibition of the Caz+-ATPaseis enhanced by iron
or iron-containing compounds such as hemoglobin and
hemin.22
Free iron and iron-containing compounds are believed to
act as catalysts in the formation of reactive oxygen species.
This is seen in the Fenton reaction, in which hydrogen
peroxide and ferrous ion generate a hydroxyl radical:
+
RYTHROCYTES O F persons with sickle cell anemia
are subject to many changes due to the polymerization of hemoglobin S . Some of the membrane changes
include: altered ion transport, altered cytoskeletal structure, phosphatidylserine transmembrane movement, and
reduced enzyme function.‘ These changes appear to be
associated with the formation of dense irreversibly sickled
cells, the hallmark of this disease.
It has been speculated that the formation of these dense
cells may be caused by a transient increase of intracellular
calcium. Indeed, alterations in Ca2++ Mg’+-ATPase activities have been shown in sickle cell membranes, particularly
that portion stimulated by ~almodulin.2.~
Sickle cell membranes are more oxidatively stressed than
normal cells. Sickle cells spontaneously generate more
reactive oxygen species including superoxide anion, hydroxyl radical, and hydrogen p e r ~ x i d eAdditionally,
.~
antioxFrom the USA Comprehensive Sickle Cell Center, University of
South Alabama, Mobile.
Submitted May 20, 1991; accepted October 22,1991.
Supported by the National Institutes of Health Grant No. P60-HL38639.
Presented in part in abstract forms at the 3lst annual meeting of the
American Society of Hematology in Boston, MA, 1990, and at the
annual meeting of the American Society of Biochemishy and Molecular Biology, New Orleans, LA,1990.
Address reprint requests to R. Blaine Moore, PhD, FAIC, USA
Comprehensive Sickle Cell Center, University of South Alabama, 2451
Fillingim St, Mobile, A L 3661 7.
The publication costs of this article were defiayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement”in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1992 by The American Society of Hematology.
0006-4971I921 7905-0003$3.00/0
1334
+
+
Fe2++ H 2 0 2+ Fe3+ OH-
+ .OH
Other iron-containing compounds may be able to replace
the free iron molecule as the Fenton reagent in this
reaction. The membranes of sickle cells possess higher
levels of denatured hemoglobins,23hemin, and nonheme
iron.Z4,25
It is speculated that the nonheme iron component
Blood, Vol79, No 5 (March 1). 1992: pp 1334-1341
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
tBHP EFFECTS OF SICKLE CELL Caz+ + Mg”-ATPase
is free iron bound to the membrane, possibly chelated to
anionic phospholipids. These iron compounds enhance the
oxidative stress in sickle cell membranes, and this has been
proposed as the cause for many of the changes that occur in
sickle cell anemia, including dehydration and adhesivity.26
Because the Caz+-ATPaseis sensitive to oxidative injury,
particularly that catalyzed by the presence of iron or
iron-containing compounds, it is proposed that the altered
CaZ’-ATPase of sickle cell membranes may be due to
iron-induced formation of reactive oxygen species. Consequently, both the sensitivity of sickle cell and normal
membranes to treatment with tBHP were examined. Protection against this loss of enzyme activity by ascorbate and
desferal was also tested.
MATERIALS AND METHODS
Preparation of eythrocyte membranes. Blood was obtained with
consent from normal and sickle cell donors. The blood was
centrifuged at 7,OOOg for 1 minute, and the plasma removed by
aspiration. The packed red blood cells (RBCs) were washed four
times with 0.9% NaCl solution. The supernatant and bum coat
were removed after each washing. White ghosts were made by
lysing 2 mL of cells with 30 mL of 10 mmol/L Tris [Tris(hydroxymethyl)-amino methane] buffer (pH 7.4) containing 1 mmol/L
EDTA (ethylenediaminetetraaceticacid). Pellets were washed
EDTA and twice more with 10
once more with Tris buffer
mmol/L Tris buffer (pH 7.4). Membrane pellets were collected
after each step by centrifuging the lysed suspension at 15,OOOg for
10 minutes. The procedure for preparing pink ghosts, ie, membranes containing hemoglobin, have been reported previously.”
The concentration of hemoglobin in these preparations was measured to be about 2 pmol/L or 4% of the total membrane protein.
Membrane protein analysis. Membrane protein was measured
routinely by the Bradford procedure*’using bovine plasma gamma
globulin as standard.
Treatment of membranes with tBHP, ascorbate, and desferal.
Suspensions of white ghosts (2 mL, 3 to 5 mg protein/&) were
incubated with 0.5 mmol/L tBHP for 30 minutes at 37°C. Ascorbate and/or desferal were present in suspensions at a final
concentration of 1 mmol/L where indicated in the figure legends.
After incubation, the samples were centrifuged at 15,OOOg for 10
minutes. Two 0.4-mL samples from each supernatant were collected for use in the malondialdehyde measurement. The ghost
pellets were washed twice with 15 vol (approximately 10 mL) of 10
mmol/L Tris buffer (pH 7.4). The final ghost pellets were resuspended to 1.0 mL with the same 10 mmol/L Tris pH 7.4 washing
buffer.
Malondialdehyde (MDA) measurement. MDA was measured by
a modification of the procedures described by Bidlack and Tappel.= To each 0.4-mL sample of supernatant or standard was added
0.85 mL of 0.47% 2-thiobarbituric acid and 0.25 mL of 100%
trichloroacetic acid. Each tube was placed in a boiling water bath
for 15 minutes to develop the color and then cooled in a
room-temperature water bath for 5 minutes. Samples were centrifuged at 80% for 15 minutes to sediment the small amount of
denatured protein. The absorbance of each sample was measured
at 532 nm in a Beckman spectrophotometer (Beckman Instruments
Tnc, Fullerton, CA). Standard MDA was prepared by acid hydrolysis of 1,1,3,3-tetramethoxypropane followed by neutralization with
NaOH. Levels are expressed in nmoles of malondialdehyde per
milligram of protein. It is understood that thiobarbituric acid
reactive products other than MDA are formed during membrane
oxidation. The results, expressed in nanomoles of MDA per
+
1335
milligram of membrane protein, should be considered as thiobarbituric acid-reactive products equivalent to reactive MDA. Total
thiobarbituric acid-reactive products associated with the membrane suspension, expressed in nanomoles of MDA per milligram
of protein, can be calculated by multiplying the supernatant MDA
values by 1.6. In the experiments using ascorbate and/or desferal,
appropriate blanks were prepared to compensate for the absorbance contributed by these compounds.
ATPase analyses. ATPase activity was measured by a modification of the method of Levine et al,29as described by Moore et al.M
In a total assay volume of 0.5 mL the following reagents were used:
imidazole buffer, 30 mmol/L, pH 7.0; NaCl, 100 mmol/L; KCI, 20
mmol/L; ouabain, 0.1 mmol/L, MgCl,, 3 mmol/L; EGTA [ethylenebis(oxyethylenenitri1o) tetraacetic acid], 0.1 mmol/L; CaCl,, 0.2
mmol/L; calmodulin, 0.1 mg/mL; ATP, 3 mmol/L. Treated erythrocyte membranes were also added to the suspension. M e ATPase was measured by omitting calcium and calmodulin. Basal
Ca2+ Mg2+-ATPasewas measured in the absence of calmodulin.
After the reaction was initiated with ATP, samples were incubated
at 37°C for 60 minutes. The reaction was terminated by the
addition of 0.7 mL ammonium molybdate reagent (0.5% ammonium molybdate, 2% sodium dodecyl sulfate, in 1N sulfuric acid).
Color was induced by the addition of 20 pL of ANSA reagent
(0.2% l-amino-2-naphthol-4-sulfonic
acid, 1.2% sodium sulfite,
1.2% sodium metabisulfite). After 30 minutes, absorbances of the
samples were read at 650 nm in a Beckman spectrophotometer.
Measurement of nonheme iron. Nonheme iron associated with
white erythrocyte membranes was measured using the ferrozine
procedure described by Kuross and HebbeLZ
3H-N-ethylmaleimide, sodium dodeql suvate-polyacylamide gel
electrophoresis (SDS-PAGE), and autoradiography. Normal and
sickle cell membranes (0.15 mL, 3 mglmL), treated with tBHP for
various times 0 to 60 minutes (Figs 1 and 2), were incubated with
0.15 mL of ’H-N-ethyl maleimide (1 mmol/L 25 cpm/pmol,
’H-NEM) for 30 minutes at 37T. The membranes were washed
twice again with 10 mmol/L HEPES [N-(2-hydroxyethyl)piperazineN’-(2-ethanesulfonic acid)] buffer pY 7.4 and then salubilized for
separation by SDS-PAGE. A 10% polyacrylamide gel was used and
electrophoresis was performed according to the procedure of
Laemmli.” The gel was stained with 0.025% Coomassie brilliant
blue in 50% methanol and 10% acetic acid. Destaining was
+
a
- - - - - - - - - --.
0.04
0
10
20
30
40
50
60
Time (min)
Fig 1. Effectsof tBHP on normal and sickle erythrocyte membrane
Ca2+ Mgz+-ATPaseand calmodulin-stimulatedCaz+ Mgz+-ATPase
activities over time. Membranes were treated with 0.5 mmol/L tBHP
as described in Mater[als an4 Methods. The values shown are means
Normal; (----),
with standard deviationsfrom three experiments. (-),
sickle 0111; (O), basal Ca2+ Mgz+-ATPase; (O), calmodulin-stimulated CaZ+-ATPase.
+
+
+
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
MOORE ET AL
1336
0.0
J
0
10
20
40
Time (min)
30
50
60
I
Fig 2. MDA formation in normaland sickle erythrocytemembranes
treated with tBHP. Membraneswere treated with 0.5 mmol/ L tBHP as
described in Materials and Methods. The values shown are means
with standard deviations from three experiments. I-),
Normal; (----I,
sickle cell; (A),MDA.
performed in 30% methanol, 10%acetic acid. The destained gel
was then scanned with an ISCO model 1312 scanner (ISCO Inc,
Lincoln, NE) and the peaks quantitated using a Spectra Physics SP
4270 integrator (Spectra Physics, San Jose, CA). To prepare the gel
for autoradiography, it was placed in “enhance solution”
(En‘Hance; NEN Research Products, Boston, MA) for 1 hour
followed by water for 30 minutes and then the gel was dried using a
slab gel drier (Model 1140; Hoefer Scientific Instruments, San
Francisco, CA). For autoradiography,the dried gel was exposed to
X A R S Kodak film (Eastman Kodak, Rochester, NY) for 10 days
at -75°C. The x-ray film was developed and the autoradiogram
peaks scanned and integrated as described above for the gel.
’H-NEM/protein ratios of membrane proteins were calculated by
dividing the integrated peak of the autoradiogram band by the
integrated peak of the Coomassie-stained gel.
RESULTS
Effects of tBHP on normal and sickle cell membranes.
Incubating a suspension of normal erythrocyte membranes
with 0.5 mmol/L tBHP for 60 minutes caused a 50%
inhibition of Ca” + Mg”-ATPase activity and a 40%
inhibition of calmodulin-stimulated Ca” + Mg”-ATPase,
respectively (Fig 1). The same treatment of sickle erythrocyte membrancs caused a basal and a calmodulin-stimulated Ca” + Mg’+-ATPase inhibition of about 75%, respectively (Fig 1).
After tBHP treatment for 60 minutes, the formation of
MDA in normal membranes increased almost 50%, while in
sickle membranes the MDA level increased almost 200%
(Fig 2).
To assess the formation of high molecular weight protein
polymers (HMWP) and protein sulfhydryl status after
oxidation, membranes were treated with ’H-NEM and the
labeled proteins separated by SDS-PAGE (Fig 3). From the
Coomassie-stained gel, it was apparent that a large band
(HMWP present at the top of the gel) composed of
polymerized membrane proteins, formed during treatment
with tBHP. The HMWP band was evident at 10 minutes
and became distinct at 30 minutes. This band formed in
both normal and sickle cell membranes but it formed more
rapidly in the latter. This was most apparent in the
NEM-labeled band on the autoradiogram for the 10-minute
samples. To dcterminc the loss of free sulfhydryl groups
due to oxidation during tBHP incubation, the amount of
’H-NEM per protein band was quantified by integrating the
peaks of the autoradiogram and the destained gel and
taking the ratio for the corresponding bands. The results
showed a 22% decrease in NEM labeling of sickle cell
bands 1 + 2 (spectrin) over the 60-minute period and a 45%
decrease in NEM labcling of sickle cell band 5 (actin)
between 30 and 60 minutes. Although some decrease in
label was observed for these bands in normal cells it was less
than that seen in sickle cells. The free sulfhydryl groups
associated with band 6, glyceraldchyde-3-phosphatedehydrogenase, were oxidized very rapidly on exposure to tBHP.
Protein Stain
.......
nMwp
Untr.
,
l+
3*
8
0
Control
10 30 60
mwm rmmmm
rqm
3H-NEM Autoradiogram
HMWP Untr.
0
1+2
L
3
4.1
4.2
Control
10 30 60
q
am.&
L.-
C
-
A
Sickle Cell
0 10 30 60
B
Sickle Cell
0 10 30 60
-===
m
Fig 3. SDS-PAGE of isolated normal and sickle cell membranes
treated with tBHP (0.5 mmol/L). From left to right: untreated control;
normal, 0 minutes; normal, 10 minutes; normal, 30 minutes; normal,
60 minutes; sickle, 0 minutes; sickle, 10 minutes; sickle, 30 minutes;
sickle, 60 minutes. HMWP, high molecular weight polymer; numbers
on the left refer to the erythrocyte membrane protein bands. (A)
Coomassie blue-stained gel; (E) an autoradiogramof the gel shown in
(A).
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
tBHP EFFECTS OF SICKLE CELL Ca2++ Mg*+-ATPase
1337
Effects of ascorbate and desferal on tBHP inhibition of
normal and sickle cell membrane Ca2+ + MgF+-ATPase.
Treatment of white erythrocyte membranes with tBHP (0.5
mmol/L) for 30 minutes did not result in significant loss of
enzyme activity or formation of MDA. Therefore, to assess
the effects of ascorbate and desferal on normal membranes,
oxidation was performed on pink ghosts. Treatment of pink
erythrocyte membranes from normal subjects with 0.5
mmol/L tBHP caused a significant decrease in both basal
and calmodulin-stimulated Caz++ MgZ+-ATPaseactivities
(63% and 68%, respectively; Fig 4A). The addition of
ascorbate to these membranes prevented the tBHPinduced losses of basal and calmodulin-stimulated activities
allowing only 35% and 30% decreases, respectively. Treatment with 1 mmol/L desferal in the presence of tBHP
yielded better protection, resulting in 22% and 14% inhibition of the basal and calmodulin-stimulated Caz+-ATPase
activities relative to controls. The greatest protection against
the loss of activity was obtained when both ascorbate and
desferal were added to the membrane suspension containing tBHP. The basal Ca” + M$+-ATPase activity decreased by only 13% of the control, while the calmodulinstimulated Ca2++ M$+-ATPase activity did not decrease at
all. In each case the addition of ascorbate, desferal, or
ascorbate + desferal resulted in ATPase activities that
were not significantly different (Student’s t-test for unpaired variates) relative to those of untreated membranes.
The ATPase activities were significantly different from the
activities of membranes treated with tBHP alone. The
production of MDA increased on treatment with 0.5
mmol/L tBHP. Treatment of the membranes with 1mmol/L
ascorbate or 1 mmol/L desferal (both in the presence of
tBHP) gave some protection against MDA production.
Again, the greatest protection was seen in the samples
treated with both ascorbate and desferal, in which only
0.382 nmol MDA/mg membrane protein were produced.
Treatment of sickle cell membranes with 0.5 mmol/L
tBHP (Fig 4B) resulted in a greater inhibition of basal and
calmodulin-stimulated Ca” + MgZ+-ATPaseactivities compared with normal membranes. In contrast to that observed
in normal membranes, the addition of 1 mmol/L ascorbate
T 2.1
,
1
>
- 1.5
w
E
1.2
2
0.9
0.6
0.6
a 0.3
0.3
nn
0.0
~~
Ctl
A
1.5
aB
y
v
p
9
W
+
4
L
E-
d
>
- 1.5
v
0.9
tBHP
tBHP
tBHP
Desf
Asc
tBHP
Asc
Desf
h
11.8
vi
1.2
f
f 1.8
11.8:
m
.-3
.c
T 2.1
h
1.5
0
v
, 2.1
and tBHP to sickle cell membranes provided little protection, ie, the ATPase activities of membranes treated with
tBHP + ascorbate were not significantly different from the
activities from membranes treated with tBHP alone but
were still highly significantly different (P < .005) from the
ATPase activities of untreated membranes. Likewise, the
use of 1 mmol/L desferal was ineffective in significantly
reducing the inhibition of the Caz+ + MgZ+-ATPaseby
tBHP. However, treatment with both ascorbate and desferal in the presence of tBHP was effective, allowing only
31% and 15% decreases in the basal Ca” + M$+-ATPase
and calmodulin-stimulated CaZ++ MgZf-ATPaseactivities,
respectively. The levels of MDA production showed significant increases in the samples treated with tBHP and in the
samples treated with ascorbate and tBHP. The levels
increased from 0.290 nmol/mg in the sickle cell control to
1.147 nmol/mg and 1.225 nmol/mg in the tBHP alone and
tBHP + ascorbate samples, respectively. Treatment with
both ascorbate and desferal reduced the MDA formation to
a greater extent than with desferal alone.
Effects of increasing concentrations of desferal on Ca2+ +
M$’-ATPase activities in sickle erythrocyte membranes.
When a suspension of sickle erythrocyte membranes was
treated with tBHP (0.5 mmol/L) and incubated for 30
minutes at 3TC, the basal Caz++ Mg2+-ATPaseactivity was
inhibited 71% (Fig 5, left) and the calmodulin-stimulated
CaZ++ MgZ+-ATPaseactivity was inhibited 70% (Fig 5,
right). Addition of 1 mmol/L ascorbate in the presence of
tBHP increased the activities minimally (0.162 pmol/mg/h
in the basal, 0.469 pmol/mg/h in the calmodulin-stimulated). Desferal was added in increasing concentrations in
the presence of tBHP. In both the basal and the calmodulinstimulated samples, the greatest increase was seen when
the membrane suspensions were treated with 1 mmol/L
desferal. The basal Caz++ Mgz+-ATPaseactivity increased
to 56% of the untreated sample and the calmodulinstimulated CaZ++ MgZ+-ATPaseincreased to 78% of the
untreated sample.
Effects of ascorbate on tBHP inhibition of normal membranes in the presence of hemoglobin and iron. We have
reportedz6 previously that iron or iron-containing com-
G
gs
w
0
1.2
1.2
yE
$
.- 0.9
0.9
g
2
0.6
0.6
$
0.3
0.3
g
E
v
YI
>
.+
TI
C
W
Q
$
0.0
0.0
Ctl
B
tBHP
tBHP
Asc
tBHP
Desf
tBHP
Asc
Desf
+
Fig 4. Effects of ascorbate and desferal on Ca2+ Mg2+-ATPaseactivity and MDAformation in pink erythrocytemembranes treated with tBHP.
Membraneswere treated with 0.5 mmol/L tBHP, 1 mmol/L ascorbate, and 1 mmol/L desferal as described in Materials and Methods. Samples
were incubated for 30 minutes at 37°C. Values shown are means with standard deviations from three experiments. (A) Normal membranes; (6)
sickle cell membranes. (0)
Basal
. Ca‘+ Mg2+-ATPase;(W), calmodulin-stimulated Ca’+ Mg”-ATPase; (B), MDA.
+
+
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1338
MOORE ET AL
I
0.5
h
2
1
\
0.4
-0a,
E,
.
h
2
+Untreated
$ +Untreated
1.0
\
$
5 0.8
E,
v
x
._
>
c
2 0.3
._
T
0.6
a
c
..->
c
a,
0
a
Ld
g 0.2
a
m
+
CL
4
+;'
0.4
I
c
._
3
-tBHP
1
4
-tBHP
+
-0
NLd0.1
0
-2 o.2
0
,
0
I
1
2
3
4
[Desferall (mM)
,
5
0
1
2
3
4
[Desferall (mM)
pounds, such as hemoglobin and hemin, can enhance the
inhibition of erythrocyte membrane Ca2++ Mg*+-ATPase
caused by tBHP. Also, ascorbate at a high concentration
(20 mmol/L) protects the membrane Caz++ Mgz+-ATPase
from oxidative damage by tBHP when hemoglobin or hemin
is present.
The effective concentrations of ascorbate that protect
against tBHP inhibition of ATPase activities and MDA
formation in white erythrocyte membranes in the presence
of 2 pmol/L hemoglobin are shown in Fig 6. Untreated
samples had basal and calmodulin-stimulated Ca2++ M$+ATPase activities of 0.66 and 1.21 Fmol/mg/h, respectively,
with minimal MDA formation (0.037 nmol/mg). The addition of 0.5 mmol/L tBHP caused a greater than 90%
reduction in ATPase activities and generated 2.36 nmol/mg
of MDA. At the lowest concentrations of ascorbate, 10 to
i
IL
Fig 5. Effects of increasing concentrations of desMg'+-ATPase activities in sickle
feral on the Ca'+
erythrocyte membranes treated with tBHP and ascorbate. Membranes were incubated at 37°C for 30
minutes with 0.5 mmol/L tBHP, 1mmol/L ascorbate,
and increasing concentrations of desferal (0.1 t o 5
mmol/L). (0).Untreated membranes; (A), tBHPtBHP-treated membranes
treated membranes; (0).
in the presence of ascorbate.
5
100 pmol/L, the major effect observed is a 77% reduction
in MDA formed. In contrast, the inhibition of the CaZ++
MgZ+-ATPaseby tBHP was not protected at all by ascorbate
over this same concentration range (0 to 100 Fmol/L).
Increasing the concentration of ascorbate from 0.1 mmol/L
to 0.5 mmol/L provided significant protection for the
Ca2++ Me-ATPase with only a 34% reduction in activity,
and reduced the MDA formation to near background levels
of 0.17 nmol/mg. From these results it was decided that 1
mmol/L ascorbate could be used in experiments as a
protective concentration against MDA formation with only
a minor loss (30%) of enzyme activity.
The effects of ascorbate on tBHP inhibition of ATPase
activities and MDA formation in white membranes plus 0.1
mmol/L FeC12-1 mmol/L adenosine diphosphate (ADP)
are shown in Fig 7. In contrast to the protective effects of
ascorbate with hemoglobin-enhanced tBHP oxidation, ascorinn
1 9
-1.5
0.0
0.2
0.4
0.6
0.8
Ascorbate Concentration (mM)
1 .o
Fig 6. Effects of increasing concentrations of ascorbate on tBHPtreated white ghosts in the presence of hemoglobin. Normal white
ghosts were incubated for 30 minutes at 37°C in the presence of 0.5
mmol/ L tBHP, 2 pmol/L hemoglobin, and increasing concentrations
of ascorbate, 0.05 t o 1 mmol/L. After centrifugation the supernatants
were assayed for malondialdehyde (thiobarbituric acid-reactive products). The remaining membrane pellets were washed twice with 10
mmol/L Tris/HCI buffer pH 7.4 and then assayed for ATPase activities. Values shown are the means of duplicate analyses from t w o
experiments: (O), basal Ca'+ + MgZ+ATPase; (O), calmodulinstimulated Ca2+ Mg'+-ATPase; (A),MDA.
+
2.0
0.2
0.0
0.0
0.2
0.4
0.6
0.8
Ascorbate Concentration (mM)
5
1 .o
Fig 7. Effects of increasing concentrations of ascorbate on tBHPtreated white ghosts in the presence of FeCI,-ADP. White membranes
were incubated for 30 minutes at 37°C in the presence of 0.5 mmol/L
tBHP, 0.1 mmol/L FeCI,, 1 mmol/L ADP, and increasing concentrations of ascorbate, 0.02 t o 1.0 mmol/L. MDA and ATPase procedures
were performed as described in Fig 6 and Materials and Methods.
Values shown are the means and standard deviations of duplicate
analyses from three experiments. (O),Basal Ca2+ Mg'+-ATPase; (0).
calmodulin-stimulated Ca"
Mg2+-ATPase;(A),MDA.
+
+
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
+ Mg2+-ATPase
1339
tBHP EFFECTS OF SICKLE CELL Ca2+
bate increased iron-enhanced tBHP oxidation of white
membranes. The basal and calmodulin-stimulated Caz+ +
M$+-ATPase activities decreased further and were all but
eliminated at ascorbate concentrations of 100 pmol/L. In
concert with this loss of enzyme activity, the formation of
MDA increased dramatically from 1.2 to 6 nmol/mg membrane protein. At 1 mmol/L ascorbate the MDA formation
neared 10 nmol/mg membrane protein, almost an order of
magnitude higher than that produced by iron and tBHP
alone with membranes.
In our previous report,” we showed that iron-enhanced
tBHP oxidation caused half-maximal inhibition of Caz+ +
M$+-ATPase activities at about 30 pmol/L FeCl,. In Fig 8
we show that ascorbate (1 mmol/L) shifts the effects of
iron-tBHP inhibition of enzyme activity and MDA formation to between 5 and 10 p.mol/L FeCI,. The concentration
range of FeCI, that produced optimal inhibition of the
Caz++ Me-ATPase activities and MDA formation was 20
to 50 pmol/L. This study was repeated with increasing
hemoglobin concentrations (50 pmol/L to 2 mmol/L) in
the presence of ascorbate and little to no change in ATPase
activities or MDA formation was observed (R.B.M., L.D.J.:
unpublished results, May 1991).
The results of Figs 7 and 8 indicate that low concentrations of ascorbate and iron can cause significant membrane
oxidation and loss of enzyme activity. For this reason we
measured the endogenous levels of nonheme iron associated with the membranes of normal subjects, subjects with
sickle cell anemia, and subjects with other sickling disorders. The results are shown in Table 1. The average
membrane nonheme iron level of sickle cell subjects was
11.75 nmol/mg membrane protein, a value significantly
higher than that of normal subjects or subjects with sickle
cell &thalassemia or SC hemoglobinopathy. These results
are similar to those published previously25 for sickle cell
subjects, considering the different membrane protein concentrations that would be obtained using gamma globulin
rather than albumin as the protein standard in the Bradford
>>
f
1.2
*
-E
.-
1.0
0.8
Sp’ thalassemia
sc
Nonheme Iron
(nmolhg)
9
14
3
2
0.07 f 0.12
11.75 2 5.30
0.16
0.13
v
0.2
2.1
1.8
1.8
h
1.5
1.5
1.2
1.2
40
60
[Feci21
80
100
PM
Fig 8. Treatment of white erythrocyte membranes with tBHP,
ascorbate, and increasing concentrations of FeCI,-ADP. White membranes were treated with 0.5 mmol/L tBHP and 1 mmol/L ascorbate
for 30 minutes at 37°C in the presence of increasing concentrationsof
FeCI,-ADP. The ratio of FeCI, to ADP was kept constant at 1/10.
Values shown are the means of duplicate analyses from three
experiments. (O), Basal Caz+
Mg2+-ATPase; (0). calmodulinstimulated Caz++ Mg’+-ATPase; (A),
MDA.
+
5
-E
0
g
: .9
0.3 8
I
0.6
C
0.3
0.0
F
25
0
0.9
P)
20
< ,0001
2.1
5 0.9
‘5
.5 0.6
0.4
0
PValue
assay. Assuming that there are about 6 mg membrane
protein per mL of ghost suspension (or per milliliter of
packed RBCs), then the average nonheme iron concentration would be about 70 pmol/L.
The decrease in iron-ascorbate-tBHP oxidation of erythrocyte membranes by desferal. Because ascorbate was observed to augment the oxidative effects on membranes by
tBHP and FeCI,, we performed experiments to determine if
chelation of the iron by desferal would alleviate the
oxidative injury (Fig 9). As expected, marked reduction in
Ca2++ MgZ+-ATPaseactivities and moderate formation of
MDA occurred when white membranes were treated with
tBHP and FeCI, (100 pmol/L) + ADP (1 mmol/L). The
addition of ascorbate to this oxidative system produced a
dramatic increase in MDA production from 1.4 to 10.4
nmol/mg protein with complete obliteration of Ca2+ +
M$+-ATPase activities. Desferal was found to be very
effective in diminishing the oxidative injury caused by
FeCl, + tBHP and by FeCI, + tBHP + ascorbate. In the
former oxidative system, ie, FeCI, + tBHP, the chelation of
iron by desferal might be expected to restore the enzyme
activities and MDA formation to those seen with tBHP
treatment of white membranes. However, the Ca” +
M$+-ATPase activities obtained with the addition of desferal were higher than expected from tBHP alone and this
observation was supported by the results for normal white
membranes presented in Fig 4A. The addition of desferal to
the iron-ascorbate-tBHP oxidative system produced remarkable protection increasing the calmodulin-stimulated activity from 0.036 to 1.033 pmol/mg/h and decreasing the
g
0.6
0
%
ss
aE
2-
c
g
AA
N
1T
QJ
:E
*
4
Hemoglobin Type
-
-
c
Table 1. Nonheme Iron Levels Associated With Erythrocyte
Membranes
o.n
on
Ctl
tBHP FetBHP FetBHP FetBHP FetBHP
Asc
Desf
A+D
+
Fig 9. Effects of tBHP, ascorbate, and desferal on Ca”
Mgz+ATPase activities and MDA formation in normal white membranes in
the absence and presence of 100 bmol/L FeCI,. White membranes
were incubated with 0.5 mmol/L tBHP at 37°C for 30 minutes in the
absence or presence of 1 mmol/L ascorbate and/or 1 mmol/L
desferal. Values shown are the means and standard deviations of
duplicate analyses from four experiments. (0).
Basal Ca2+ Mg*+AtPase; (m), calmodulin-stimulated Caz+ Mg”-ATPase; (a),
MDA.
+
+
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
MOORE ET AL
1340
MDA production from 10.4 to 0.16 nmol/mg. Although not
shown, the presence of ascorbate and iron without tBHP
caused about a significant decrease (>50%) in both the
basal and calmodulin-stimulated Caz+
Mg2+-ATPase
activities that could be prevented by the addition of
desferal.
+
DISCUSSION
The effects of tBHP treatment on sickle cell membranes
were enhanced relative to those on normal white erythrocyte membranes. In sickle cell membranes this treatment
causes significant loss of Caz+ M$'-ATPase activity and
is accompanied by an increase in the formation of MDA, a
thiobarbituric acid-reactive product of lipid peroxidati~n.'~.'~
Because the loss of enzyme activity was greater and the
MDA formation levels were higher in sickle cell membrane
samples than in normal membrane samples treated with the
same concentration of tBHP (0.5 mmol/L), it became
evident that some membrane-associated component of the
sickle cell membrane may be acting to catalyze the oxidative
effects of tBHP. It has been shown that many iron or
iron-containing compounds are found in excess levels in
sickle cells,23 and it is also known that some forms of
physiologic iron can catalyze the formation of reactive
oxygen species through the Fenton reaction. There is even
visible evidence of some membrane component present in
sickle cells that is not seen in normal cells. After identical
washings and lysings (see Materials and Methods), sickle
membranes have an amber color, while normal membranes
appear white. Based on these findings and observations,
one possible cause that was considered was that nonheme
iron and/or iron-containing components found in excess
levels in the sickle cell membrane catalyze the formation of
reactive oxygen species in the presence of tBHP, thereby
increasing oxidative damage to the cell.
The presence of a membrane-associated iron compound
has apparently been demonstrated by the effects of treatment of normal and sickle membranes with 1 mmol/L
ascorbate in the presence of tBHP. Ascorbate is known to
act as an antioxidant,22and sickle cell patients have been
shown to have lower physiologic levels of ascorbic acid and
other natural antioxidants such as vitamin E and ribofla~ i n . 6However,
.~
it has also been shown that in levels higher
+
than physiologic (20 kmol/L), ascorbate can react with free
iron molecules to catalyze the formation of hydroxyl radic a l ~ . ~ *In- ' ~our experiment, membrane suspensions were
treated with 1 mmol/L ascorbate and 0.5 mmol/L tBHP,
and incubated for 30 minutes at 37°C. In normal membranes, ascorbate showed protective effects by increasing
enzyme activities and decreasing MDA levels relative to
samples treated with tBHP alone. In sickle cell membranes,
addition of ascorbate gave different results. The enzyme
activity was increased only slightly and the MDA levels
were also increased over the levels observed in the samples
treated with tBHP alone. The ascorbate did not protect
against lipid peroxidation as it did in the normal membranes. We propose that this is due to an iron component in
the sickle cell membrane that reacts with the ascorbate to
form hydroxyl radicals and enhance oxidative injury.
If some nonheme iron is bound to the membranes of
sickle cells that can react with ascorbate to enhance
oxidation, then it is possible that chelation of this iron
component with desferal would allow ascorbate to have the
same protective effects in sickle cell membranes as it has in
normal cell membranes. Indeed, the desferal did have this
effect. Furthermore, it seemed reasonable to presume that
if a suspension of membranes were treated with both
ascorbate and desferal in the presence of tBHP, the
desferal would chelate the nonheme iron component, and
the ascorbate would be able to act as an anti-oxidant
instead of a pro-oxidant. The results support this presumption.
On treatment of normal membranes with 1 mmol/L
ascorbate and 1 mmol/L desferal in the presence of tBHP,
almost total recovery of basal Caz++ Mg2+-ATPaseactivity
was observed; a recovery of calmodulin-stimulated ATPase
activity to a level higher than the control sample not treated
with tBHP was also observed. In sickle cell membranes,
treatment with ascorbate and desferal in the presence of
tBHP gave significantly higher activities for both the basal
and calmodulin-stimulated ATPase and significantly lower
levels of MDA production than in the samples treated with
ascorbate and tBHP alone. This would support the concept
that the use of ascorbate and desferal together would help
protect against oxidative damage seen in sickle cell patients,
but further investigation must be made to assess any future
therapeutic value that these agents may have.
REFERENCES
1. Moore RB: Pathophysiologyof the sickle cell, in Mankad VN,
Moore RB (eds): Sickle Cell Anemia: Pathophysiology, Diagnosis
and Management, New York, NY, Praeger Scientific, 1992 (in
press)
2. Dixon E, Winslow RM: The interaction between (Ca2++
Me)-ATPase and the soluble activator (calmodulin) in erythrocytes containing haemoglobin S. Br J Haematol47:391,1981
3. Litosch I, Lee KS: Sickle red cell calcium metabolism: studies
on Caz+ + M$'-ATPase and Ca-binding properties of sickle red
cell membranes. Am J Hematol8:377,1980
4. Gopinath RM, Vincenzi FF: (Ca2++ Me)-ATPase activity
of sickle cell membranes: Decreased activation by red blood cell
cytoplasmic activator. Am J Hematol7:303,1979
5. Hebbel RP, Eaton JW, Balasingam M, Steinberg MH: Spontaneous oxygen radical generation by sickle erythrocytes. J Clin
Invest 70:1253,1982
6. Jain SK, Williams DM: Reduced levels of plasma ascorbic
acid (vitamin C) in sickle cell disease patients: Its possible role in
the oxidant damage to sickle cells in vivo. Clin Chim Acta 149:257,
1985
7. Natta C, Stacewicz-Sapuntazakis M, Bhagavan H, Bowen P:
Low serum levels of carotenoids in sickle cell anemia. Eur J
Haematol41:131,1988
8. Varma RN, Mankad VN, Phelps DD, Jenkins LD, Suskind
RM: Depressed erythrocyte glutathione reductase activity in sickle
cell disease. Am J Clin Nutr 38:884, 1983
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
tBHP EFFECTS OF SICKLE CELL Ca2+ + MgZ+-ATPase
9. Adelekan DA, Thurnham DI, Adekile AD: Reduced antioxidant capacity in pediatric patients with homozygous sickle cell
disease. Eur J Clin Nutr 43:609,1989
10. Jain SK, Shohet SB: A novel phospholipid in irreversibly
sickled cells: Evidence for in vivo peroxidative membrane damage
in sickle cell disease. Blood 63:362,1984
11. Rank BH, Carlsson J, Hebbel R P Abnormal redox status of
membrane-protein thiols in sickle erythrocytes. J Clin Invest
75:1531,1985
12. Jain SK, Shohet S B Calcium potentiates the peroxidation of
erythrocyte membrane lipids. Biochim Biophys Acta 64246,1981
13. Jain S K The accumulation of malondialdehyde, a product of
fatty acid peroxidation, can disturb aminophospholipid organization in the membrane bilayer of human erythrocytes. J Biol Chem
259:3391,1984
14. Hochstein P, Jain SK: Association of lipid peroxidation and
polymerization of membrane proteins with erythrocyte aging. Fed
Proc 40183,1981
15. Moore RB, Brummitt ML, Mankad V N Hydroperoxides
selectively inhibit human erythrocyte membrane enzymes. Arch
Biochem Biophys 273:527,1989
16. Davies KJ, Goldberg AL: Oxygen radicals stimulate intracellular proteolysis and lipid peroxidation by independent mechanisms in erythrocytes.J Biol Chem 2628220,1987
17. Graf E, Verma AK, Gorski JP, Lopaschuk G, Niggli V,
Zurini M, Carafoli E, Penniston JT: Molecular properties of
calcium-pumpingATPase from human erythrocytes. Biochemistry
21:4511,1982
18. Hebbel RP, Shalev 0, Foker W, Rank BH: Inhibition of
erythrocyte Ca*+-ATPaseby activated oxygen through thiol- and
lipid-dependent mechanisms. Biochim Biophys Acta 8628,1986
19. Scutari G, Ballestrin G, Cavaz AL: Diavalent cation dependent ATPase activities of red blood cell membranes: Influence of
the oxidation of membrane thiol groups close to each other. J
Supramol Struct 14:1,1980
20. Niggli V, Adunyah ES, Carafoli E: Acidic phospholipids,
unsaturated fatty acids, and limited proteolysis mimic the effect of
calmodulin on the purified erythrocyte Caz+-ATPase.J Biol Chem
25623588,1981
21. Snider RL, Moore RB: The role of lipid peroxidation in
tert-butylhydroperoxide inhibition of human erythrocyte calcium + magnesium ATPase. Bios 5765,1988
1341
22. Moore RB, Bamberg AD, Wilson LC, Jenkins LD, Mankad
V N Ascorbate protects against tert-butyl hydroperoxide inhibition
of erythrocyte membrane Ca2+ + Me-ATPase. Arch Biochem
Biophys 278:416,1990
23. Asakura T, Minakata K, Adachi K, Russell MO, Schwartz E
Denatured hemoglobin in sickled erythrocytes. J Clin Invest
59633,1977
24. Kuross SA, Rank BH, Hebbel RP: Excess heme in sickle
erythrocyte inside-out membranes: Possible role in thiol oxidation.
Blood 71:876,1988
25. Kuross SA, Hebbel RP: Nonheme iron in sickle erythrocyte
membranes: Association with phospholipids and potential role in
lipid peroxidation. Blood 72:1278,1988
26. Hebbel RP, Ney PA, Foker W: Autoxidation, dehydration,
and adhesivitymay be related abnormalities of sickle erythrocytes.
Am J Physio1256:C579,1989
27. Bradford M: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal Biochem 72:248,1976
28. Bidlack WR, Tappel AL: Damage to microsomal membrane
by lipid peroxidation. Lipids 8:177,1983
29. Levine SN, Berkowitz LR, Orringer EP: Cetiedil inhibition
of calmodulin-stimulated enzyme activity. Biochem Pharmacol
33:581,1984
30. Moore RB, Manery JF, Still J, Mankad VN: The inhibitory
effects of polyoxyethylene detergents on human erythrocyte acetylcholinesterase and Ca2+ + Mg2+-ATPase. Biochem Cell Biol
67:137,1989
31. Laemmli UK Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680,1970
32. Rowley DA, Halliwell B: Formation of hydroxyl radicals
from hydrogen peroxide and iron salts by superoxide-and ascorbatedependent mechanisms: Relevance to the pathology of rheumatoid
disease. Clin Sci 64649,1983
33. Winterbourn C C Comparison of superoxide with other
reducing agents in the biological production of hydroxyl radicals.
Biochem J 182:625,1979
34. Miller DM, Aust S D Studies of ascorbate-dependent,
iron-catalyzed lipid peroxidation. Arch Biochem Biophys 271:113,
1989
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1992 79: 1334-1341
Increased susceptibility of the sickle cell membrane Ca2+ + Mg(2+)ATPase to t-butylhydroperoxide: protective effects of ascorbate and
desferal
RB Moore, TM Hulgan, JW Green and LD Jenkins
Updated information and services can be found at:
http://www.bloodjournal.org/content/79/5/1334.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.