From www.bloodjournal.org by guest on April 28, 2018. For personal use only.

Sucrose Density Gradient Analysis of Erythrocyte

Membranes in Hemolytic Anemias

By Thomas P. Flynn, Gerhard J. Johnson, and David W. Allen

To investigate the membrane abnormalities that may play phosphate dehydrogenase (G6PD) variants. Dense mem-
a pathophysiologic role in several hemolytic anemias we brane subpopulations found in RBC membranes from three
determined the density distribution on sucrose density splenectomized patients with Hb KIn were associated
gradients of human red blood cell (RBC) membranes from with adsorbed globin. while similar subpopulations in RBC
patients with these disorders, from normal controls, and membranes from three splenectomized patients with
from incubated normal RBC. We analyzed the fractions for hereditary spherocytosis demonstrated increased SG due
membrane-adsorbed hemoglobin (Hb). globin. and nonglo- to adsorbed Hb. Splenectomized normals had no such
bin cytoplasmic proteins. The relationship between the abnormality in membrane density. Sucrose density
cytoplasmic proteins adsorbed on the membranes and the gradients demonstrate that membrane bound cytoplasmic
specific gravity (SG) of the membranes was linear. An protein is characteristic of the RBC membranes in several
increase in SC of the entire membrane population was hemolytic disorders. Additionally. gradients are useful for
seen in Hb C disease due to adsorbed Hb. Subpopulations the isolation and further analysis of those subpopulations
of membranes with increased SG due to adsorption of of RBC membranes with abnormal SG and exaggerated
nonglobin protein were evident in the membranes from membrane protein abnormalities.
two splenectomized patients with hemolytic glucose-6-

A LTHOUGH extensively studied with RBC incu- branes to determine the relationship of membrane-
bated under a variety of conditions in vitro, bound protein to membrane density.
increased RBC membrane binding of cytoplasmic
MATERIALS AND METHODS
proteins has not been systematically analyzed in
hemolytic anemias. Weed and coworkers’ observed Both normal healthy human subjects and patients with a variety
of hemolytic anemias were studied. G6PD Long Prairie and G6PD
that when RBC were incubated aerobically in vitro
“Tomah” are previously described variants with chronic hemolytic
without added glucose there was an increased adsorp-
disease.’#{176} Hereditary spherocytosis patients had typical peripheral
tion of both Hb and non-Hb proteins to the membrane blood morphology, and abnormal unincubated osmotic fragility
with increased viscosity, and decreased deformability curves. Hb C patients had the diagnosis confirmed by isoelectric
and presumably survival. Sears et al.2 incubated RBC focusing. Hb K#{246}lnhad characteristic isoelectric focusing, heat
instability, isopropanol instability, and increased oxygen affinity.
in phosphate buffered saline (PBS) and showed an
Blood was obtained aseptically, white blood cells and platelets
increase in globin content of the membrane on polya-
were removed by centrifugation, and RBC washed with phosphate
crylamide gel electrophoresis in sodium dodecyl buffered saline pH 7.4, and membranes prepared as
sulfate (PAGE SDS). Numerous workers have except that the hemolyzing and wash solutions (5 mM phosphate
analyzed RBC membrane polypeptides3 with PAGE pH 8) contained 1 mM EDTA. RBC were quantitated by a Coulter
counter and by microhematocrit. In certain experiments, washed
SDS in model systems and various hemolytic anemias.
RBC were aerobically incubated in 2 volumes of Dulbecco’s PBS
A distinctive pattern of changes was observed due, in
without glucose or calcium at pH 7.4, 37#{176}C
for 16-24 hr. In other
part, to increased recovery of extrinsic membrane experiments, washed RBC were incubated in 5 volumes of PBS with
proteins and to membrane-adsorbed cytoplasmic acetylphenylhydrazine (I mg/mI), 90 mm, 37#{176}C
or in 9 volumes of
proteins which included globin,2 and bands 4.2, 4.5 Tris buffered saline pH 8 for 90 mm, 37#{176}Cwith 0.2-0.8 mM
diamide. Membrane protein,’3 Hb by the o-tolidine method,’4 lipid
(catalase), 5, and 6 (glyceraldehyde-3-phosphate
phophoru5,IS6 PAGE 52.I7 and membrane polypeptides5 were
dehydrogenase).7 However, membranes lose extrinsic
analyzed as before. Membrane spectra were obtained on a Gilford
membrane proteins as well as Hb on the successive 250 spectrophotometer.
washes needed for isolation. Further, there has been no Membrane SG was measured on 0.2-0.5 ml (1-3 mg protein) of
clear indication of whether changes in membrane
composition are uniform or confined only to subpopu-
lations.
From the Hematology Section. Department of Medicine, Univer-
Sucrose density gradients have been used to analyze sity of Minnesota Medical School and Veterans Administration
Hb-binding of membranes8 and to separate mem- Medical Center. Minneapolis. Minn.
branes containing phenylhydrazine-induced Heinz Supported in part by the Veterans Administration. NIH 2POl-

bodies.9 In this work, we have isolated RBC HL-16833-06 and 5-T-32 HL-07062.
Submitted July 10, 1980; accepted August 29. 1980.
membranes from incubated RBC and several hemo-
Address reprint requests to David W. Allen, M.D.. Hematology
lytic anemias on sucrose density gradients as a repro- Section (1 1 IE), V.A. Medical Center, Minneapolis, Minn. 554! 7.
ducible method of membrane preparation and used the 8 1981 by Grune & Stratton, Inc.
polypeptide composition of the fractionated mem- 0006-4971/81/5701-0012$01.00/0

Blood, Vol. 57, No. 1 (January), 1981 59

 From www.bloodjournal.org by guest on April 28, 2018. For personal use only.

60 FLYNN. JOHNSON, AND ALLEN

once washed membranes placed on 32 ml I 5%-60% linear sucrose 600

density gradients in 5 mM phosphate 1 mM EDTA, pH 8 and
centrifuged 75,000 g for I 6 hr at 5#{176}C
in a preparative ultracentri-
fuge. Density equilibrium was reached after 4 hr. One ml fractions
were collected, adsorbance was measured at 280 nm, and SG
obtained initially by weighing 0.5 ml volumes and more recently 500
equivalent results were obtained by using a temperature-controlled
(20#{176}C) Abb#{233}3L refractometer (Bausch and Lomb, Rochester,
A
N.Y.). Measurement of the SG of 20 sucrose gradient samples both
by weighing (to the nearest 0.1 mg, or 4 significant figures) and by
the refractive index (to 5 significant figures) agreed within the 400
U)
nearest .001 SG units (4 significant figures) and are reported as 0
such. Measurement with the refractometer did not require accuracy
in filling a pipette, provided precise temperature control, and was C
U)
considerably more rapid. The turbidity imparted by the membranes 4-

to the sucrose solutions did not interfere with measurement of the

0 300
refractive index and the linearity of the gradient was maintained
throughout sample collection. Membrane SG was taken as that of 0
U)
0
the median fraction obtained graphically from a cumulative (inte- 0
grated) plot of the adsorbancy versus SG (See Fig. 2 or Fig. 6 at 0
U)
200
50% of total). The gradient fractions were diluted with 10 volumes 0
of 5 mM phosphate I mM EDTA, pH 8, and centrifuged at I 05,000
g for 60 mm for analysis of the membrane pellets by PAGE SDS.
For the hemoglobin binding experiments’8 membranes were
prepared from normal controls and incubated with 0.2-20 pM Hb
A, 5, and C in S mM phosphate at pH 6.2 for 1 5 mm at 20#{176}C
prior
to centrifugation and analysis. At this lower pH considerable Hb
A0
binding occurred’8 (see below). In other experiments, such
membranes were separated on sucrose gradients as above.
When at least three observations are available the results are
presented as the mean ± SD. Standard statistical methods were 1.15 1.16 117 118 1.19 1.20 1.21 1.22
Specific Gravity
RESULTS
Fig. 1 . Adsorbed cytoplasmic protein (fg/erythrocyte mem-
Sucrose density gradients provided a method of
brane) (ordinate) versus specific gravity (abscissa). Membranes
isolating erythrocyte membranes in a reproducible from normal erythrocytes incubated in vitro as follows: aerobical-
procedure that could be used to define membrane- ly. without glucose. 22 hr (C); with 0.8 mM diamide (A); with
acetylphenylhydrazine (1 mg/mI) (U). Membranes from normal
bound cytoplasmic protein. Proteins, such as Hb, that
controls (0) and patients with hemoglobin K#{246}ln
(a). hemoglobin
can be washed off the membranes were almost CC disease (X). hereditary spherocytosis (0 ), G6PD mutants with
completely removed. Thus, when normal fresh control chronic hemolytic disease (+ ). The equation of the line is:

membranes were incubated with 1-10 j.tM Hb at pH adsorbed protein = - 8572 + 741 4 (SG).

6.2 in 5 mM phosphate buffer, and then separated by

centrifugation without changing the pH or Hb concen-
tration’8 they contained 43.3 ± 4.1% globin by PAGE The regression line drawn through the 26 points (open
SDS (which did not distinguish Hb and globin). If the symbols) has an r value of 0.93 (p < .0005), indicating
same membranes with Hb bound at pH 6.2 were a highly significant positive correlation between
separated on sucrose gradients at pH 8 (see Materials adsorbed membrane protein and specific gravity.
and Methods), the globin content was reduced to I .3% The amount of adsorbed protein was calculated
(n = 4). Thus, we defined adsorbed membrane protein from the membrane composition as follows. The frac-
as that additional membrane-bound protein not tion fofadsorbed cytoplasmic protein was obtained by
removed by sucrose gradient isolation of the planimetry of the PAGE SDS analysis of the sucrose
membranes. Forty-eight sucrose gradients of I2 gradient fractions. The adsorbed cytoplasmic protein,
normal controls showed SG = I . I 5 1 ± .003, Hb = x, (globin and nonglobin) was assumed added to the
1.1 ± .4%. 500 fg of normal membrane protein per cell already
Figure I demonstrates the relationship between the present. Thus, f = x/(500 + x) or x - 500 f/( I f). -

amount of adsorbed cytoplasmic protein to the SG of By utilizing model systems in which large amounts
the RBC membranes from all the hemolytic anemias of membranes were available for replicate analysis we
studied, in which such measurements are available. confirmed the validity of this relationship (Table I).
For simplicity, only two normal controls were plotted. In these systems adsorbed cytoplasmic protein was
 From www.bloodjournal.org by guest on April 28, 2018. For personal use only.

RBC MEMBRANE DENSITY IN HEMOLYTIC ANEMIA 61

Table 1 . Relation of Membrane Specific Gravity to Adsor bed Cytoplasmic Prot em (results ± SD . n - 4)

. Diamide
Aerobic
Fresh Incubation 0.8 mM
Control (22 1w) Control D,amide

Total nonhemoglobin membrane protein per cell

(fg) 495 ± 30 561 ± 11’ 434 ± 57 513 ± 39
Adsorbed nonhemoglobin cytoplasmic protein (fg)
percell 0 66 0 79
Adsorbed hemoglobin per cell (fg) 2.5 65 2 11
Total cytoplasmic protein adsorbed (fg) 2.5 13 1 2 90
Lipid phosphorus (fg/cell) 1 1.6 ± 1.8 7.8 ± 1.4 10.3 ± 1.4 10.2 ± .8
Membranespecificgravity 1.150 ± .004 1.185 ± .008 1.148 ± .004 1.179 ± .005

‘Significantly different from appropriate control, p < 0.05.

measured by determining the difference in the protein diamide, a permeable oxidant that decreases RBC
content of membranes between incubated and control deformability and oxidizes sulfhydryls to disulfides,22
RBC. This permitted an independent estimate of produced membranes with increasing density. Figure
adsorbed cytoplasmic protein (Fig. I, solid symbols). 2 demonstrates that the adsorption of nonglobin
An analysis of covariance’9 showed no significant proteins (Table 1 ) was associated with a shift of the
difference in the value of the adsorbed protein between entire population of membranes towards increased
the hemolytic anemias and the model systems after 5G. Unlike aerobic incubation, the diamide incubation
adjustment for the within groups regression coeffi- did not significantly decrease lipid phosphorus (Table
cient. In Table 1, the approximately equal contribu- 1 ),indicating that membrane loss of lipids does not
tion of nonglobin and globin cytoplasmic proteins to explain the increased membrane density with this
the adsorbed protein is evident in the RBC incubated oxidant stress. In Figure 2, the results are plotted as
for 22 hr in PBS. With diamide treatment of the RBC, the cumulative percent of the total adsorbancy of the
seven times more of the adsorbed cytoplasmic protein membranes, an integrated plot permitting better esti-
was non-globin than globin (Table I ). Globin or mation of the SG corresponding to a median
nonglobin protein adsorbed on the membrane
produced approximately equal increased in the
specific gravity, since points representing the mem-
branes of diamide incubated RBC (dense because of
nonglobin cytoplasmic protein) and points of dense
membranes from aerobically incubated RBC (con-
taming both globin and nonglobin proteins) both lay
near the regression line (Fig. I).
Several experiments were performed to explore 0
possible causes of increased membrane density. Fresh
and
on dextran
and
strated
conditions,
aerobically-incubated

then hemolyzed
membranes
denser
gradients

RBC did
into
to yield
of identical
not yield
normal
dense

denser
and
RBC

membranes,
5G. Thus,
light
fractionated
fractions,2#{176}

under
demon-
these
I
membranes. RBC dehydrated in 5% sodium chloride
also did not result in dense membranes. Membranes
from RBC incubated in sodium based Hank’s
balanced salt solutions in the presence of the ionophore
A 231872 were dense (SG = 1.176 ± .001, n = 4)
1.21 1.20 I.I9 1.18 1.17 1.16 1.15 1.14 1.13 1.12
from adsorbed globin and nonglobin cytoplasmic
proteins. These changes were nearly prevented by Specific Gravity
blocking of the Gardos effect with substitution of
Fig. 2. Density distribution of control membranes (average of
potassium-based balanced salt solution in the incuba-
1 2 determinations) (-) and membranes from red cells incubated
tion2’ (membrane SG = I .1 55 ± .001 , n = 4). Incuba- with diamide: 0.2 mM (0), 0.4 mM (#{149}).and 0.8 mM (0). A
tion of normal RBC with increasing concentrations of cumulative plot is used.
 From www.bloodjournal.org by guest on April 28, 2018. For personal use only.

62 FLYNN, JOHNSON, AND ALLEN

G6PD- and nonglobin. Membranes from the fresh RBC of the

hemolytic mutants G6PD “Tomah” and G6PD Long
I00 Prairie have been analyzed eight times with an aver-
age percent of total membranes of the subpopulation,
90
SG > 1.165, of 9.0 ± 2.2% (fresh normal RBC
>
U
C
80 membranes 1.6 ± l.4%p < 0.005).
0
Figure 5 shows the density distribution of RBC
0
70
membranes from a splenectomized patient with hered-
U,
. itary spherocytosis. A total of 6 such determinations
60
on three splenectomized patients with this disease have
E
50 been done. The average percent of the shoulder, SG >
E

‘C 1.165, is 8.3% ± 1.2%, significantly different from

0
40 normal control membranes (0.2% ± .3%) (p < 0.005).
4- Unlike the hemolytic G6PD mutants, the increased
C
a) 30 membrane adsorption of globin alone accounts for the
U

20 increased density of this subpopulation. Three

nonsplenectomized patients with hereditary spherocy-
10 tosis (one the son of the patient shown) have no

I .20 1.19 1.18 1.17 1.16 1.15 1.14 1.13 1.12 1.11 AB
Specific Gravity

Fig. 3. Density distribution of membranes from fresh red cells

of 12 normals (--). from five samples of normal red cells
incubated for 22 hr ( . . . . ) and from fresh red cells of a patient
with G6PD ‘Tomah” (.-o-).

3
membrane fraction, and allowing better comparison of
the density distribution under different conditions. 4.1
Acetylphenylhydrazine treatment of normal RBC 4.2
produced a single population of dense, Heinz-body
containing globin-rich membranes (SG 1.161 ± .002,
15.I%globin, n = 4).
The background gained from the study of model
systems was used to analyze the membranes of hemo- 5
lytic anemias. The averaged density distribution of
fresh membranes from 1 2 normal controls, and from 5
incubations of normal RBC for 22 hr, are compared 6 o.&m
with the density distribution of membranes from fresh
RBC of a patient with G6PD “Tomah” in Fig. 3. This 7
plot (percent maximum absorbancy) allows better
analysis of distinct peaks or subpopulations, since any
decrease in absorbancy between subpopulations is
recorded as such. The membranes of the hemolytic
G6PD mutant contain a dense subpopulation between
SG of I 165 . and I . I 9. When fractions from this __+

subpopulation were analyzed by PAGE SDS, 2% of

Hb
the total membrane protein was globin and 4% was
nonglobin cytoplasmic proteins including bands
between 4.2 and 5, bands 5, 6, 7 and a new band before Fig. 4. Photograph of a portion of a slab gel using the
globin (Fig. 4). Thus, this subpopulation of G6PD discontinuous buffer system of Laemmli’7 with a 4% acrylamide
stacking gel and a 10% acrylamide running gel. Bands are
membranes like the entire population of membranes
number according to Steck.3 Normal control A. G6PD Long
from aerobically incubated normal RBC owes its Prairie. B. Arrows indicate additional bands in the membranes
increased density to adsorbed proteins, both globin from G6PD Long Prairie.
 From www.bloodjournal.org by guest on April 28, 2018. For personal use only.

RBC MEMBRANE DENSITY IN HEMOLYTIC ANEMIA 63

0- Norm studied and found to be not significantly different

A-.- 14$
from normal (SG 1.151 ± .002, and SG 1.147 ± .001,
100 n = 4), with no subpopulations of dense membranes.
One patient had his spleen removed after trauma
90 (splenectomized normal, Fig. 5), and one during the
course of gastric surgery. Both had no spleen by
80 liver-spleen scan, and peripheral blood findings of the
hyposplenic state.
.1 70
RBC
uniformly
membranes
increased
from
in density
Hb
(SG
C patients
1.163 ±
were
.006,
60 =

n = 4) (Fig. 6). This increase in density was due to the

I
50 presence of adsorbed Hb C as demonstrated by PAGE
SDS and isoelectric focusing of the hemoglobin from
40 the membranes. The Hb C on the membranes had the
spectrum of oxyhemoglobin, with maxima at 540 and
30 580 nm. Recombination experiments with normal
membranes demonstrated that the increased adsorp-
20
tion of Hb C was a property of the Hb. With 0.2-20
10 uM Hb A or C and normal membranes on the average
2.3 x 106 more Hb C molecules than Hb A molecules
0
-r bound per RBC membrane (p < 0.005).
1. 1.19 1.18 1.17 1.16 1.15 1.14 1.13 1.12 1_Il
DISCUSSION
Specific Gravity
Despite the multiple etiologies of the hemolytic
Fig. 5. Density distribution of red cell membranes from a
anemias studied, this investigation demonstrates that,
splenectomized patient with hereditary spheroctyosis (--A--).
compared with a simultaneously determined density distribution in each, increased membrane density is proportional to
of a splenectomized normal (-0-). The points from two the adsorption on the RBC membranes of cytoplasmic
gradients of each individual are pooled.
proteins (Fig. I ). Thus, increased membrane density
correlates with increased membrane binding of protein
evidence of a dense subpopulation of cells. The dense
membranes of hereditary spherocytosis have the spec- 100 8
trum of the adsorbed oxyhemoglobin. The presence of 0

Hb is confirmed by the near equivalence of measure- 90

ment of adsorbed Hb by the o-tolidine test’4 and of

0
adsorbed globin by PAGE SDS.5 80
When the membranes from three splenectomized
70 0
patients with Hb K#{246}lnwere analyzed on sucrose
0 0
gradients two membrane peaks were present. The 60
denser of the two peaks (SG = 1 .2 1 1 ± .003, n = 6)
contained Heinz bodies by phase microscopy. The C 50
U,
second peak had a normal density (SG I 1 50 = . ± .005, U 0

n = 6) and no Heinz bodies. The entire increase in 40

density of the Heinz body-containing membranes was
30
due to their content of globin. The color of the Heinz 0

body-containing membranes was tan rather than pink 20

and the spectrum was that of a ferrihemichrome as
previously described.23 Globin assayed by PAGE SDS 10
0
exceeded Hb measured by the o-tolidine method by a
factor of 8-10, an observation which implies heme
I_O 1.19 1.18 1.17 I.16 1.15 I.I4 1.13 1.12 1.11
1055.24 Three affected relatives of the Hb K#{246}ln patients
with intact spleens had no Heinz bodies and no dense Specific Gravity
subpopulations of membranes.
Fig. 6. Density distribution of red cell membranes on four
The RBC membranes of two hematologically gradients from two patients with Hb CC disease (0) compared to
normal individuals who had been splenectomized was Hb AA (----). A cumulative plot is used.
 From www.bloodjournal.org by guest on April 28, 2018. For personal use only.

64 FLYNN, JOHNSON, AND ALLEN

whether the protein is oxidized, denatured heme-poor Increased binding of Hb C to membranes has been
globin as in Hb K#{246}ln,or oxyhemoglobin in Hb CC noted by other investigators.27 The affinity of
disease and hereditary spherocytosis, or non-Hb cyto- membranes for Hb 5, C and A2 increases with the
plasmic protein as in G6PD mutants. We hypothesize increasing positive charge of the Hb.27 The fact that
that membrane adsorption of protein may indicate increased membrane-bound protei ns characterize the
membrane damage in these hemolytic anemias and be other hemolytic anemias suggests that the membrane-
expressed in increased membrane density as well as binding of Hb C may play an etiologic role in that
decreased RBC deformability, filtration, and survival. disease, as an explanation for previous observations
In hemolytic G6PD mutants and in the two model concerning the rheologic properties of the Hb CC
systems of oxidant stress, aerobic incubation or diam- RBC.28
ide treatment of normal RBC, the increase in In the model systems and hemolytic anemias stud-
membrane protein is largely due to increased nonglo- ied here, the adsorbed membrane protein responsible
bin cytoplasmic proteins adsorbed to the membrane. for increased membrane density correlates closely with
In all three there is decreased reduced glutathione, decreased RBC deformability and survival observed
increased membrane disulfide-linked polypeptide ag- by many investigators. Thus, decreased RBC defor-
gregates, decreased deformability, and decreased mability is seen in aerobically incubated RBC’ and
survival in vivo.’#{176}’22’25
How the membrane sulfhydryl RBC treated with A 23187 and Ca in sodium-rich
oxidation’#{176}’25 is related to increased membrane protein isotonic buffers but not in potassium-rich isotonic
adsorption is not clear. buffers.2’ Decreased RBC deformability and survival
That sucrose gradients provide an efficient method are both seen in diamide treated dog RBC,25 in G6PD
of separating Heinz body-rich membranes has been mutants with chronic hemolytic disease,’#{176} hereditary
observed before.9 Thus, it is not surprising that they spherocytosis, Hb K#{246}ln,and Hb CC disease.28’29
are also successful in separating the Heinz body- Sucrose density gradients are a sensitive method of
containing membranes from RBC of splenectomized detecting membranes with adsorbed cytoplasmic
patients with Hb K#{246}ln.Although oxidant stress- protein. It is not known whether this adsorbed protein
induced damage occurs in membranes in Hb K#{246}ln directly inhibits membrane fluidity and decreases
disease (decreased glutathione, increased disulfide RBC deformability and survival, or whether the
bonds and aggregates26) any increase in membrane adsorption of cytoplasmic protein is an epiphenome-
density due to adsorbed nonglobin proteins is negligi- non reflecting other more significant changes in the
ble compared to the massive membrane adsorption of configuration of membrane proteins. Despite this
globin-containing Heinz bodies. current limitation of our understanding, sucrose
The observation that subpopulations of dense gradients detect small populations of membranes in
membranes are only evident in splenectomized which large changes have occurred, and provide a
patients with Hb K#{246}lnand hereditary spherocytosis method for the isolation and further study of such
and not in nonsplenectomized relatives with these membranes. That these subpopulations belong to
diseases, suggests that RBC containing such dense effete, prehemolytic RBC seems likely, but remains to
membranes might be sequestered and destroyed in be proven.
patients with intact spleens.

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I I . Dodge iT, Mitchell C, Hanahan Di: The preparation and 21. Dreher KL, Eaton JW, Kuettner iF, Breslawec KP, Black-
chemical characteristics of hemoglobin-free ghosts of human shear PL, White JG: Retention of water and potassium by erythro-
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 From www.bloodjournal.org by guest on April 28, 2018. For personal use only.

1981 57: 59-65

Sucrose density gradient analysis of erythrocyte membranes in hemolytic

anemias
TP Flynn, GJ Johnson and DW Allen

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