J. Mol. Biol.

(1971) 58, 775-781

Human Erythrocyte Membranes : Specific Labelling of

Surface Proteins
MARK S. BRETSCRER
Me&al Research Council Laboratory of Molecular Biology
Hills Road, Cambridge CB2 2QH, England

(Received 4 September 1970)

A very reactive, highly radioactive (about 10 Ci/m-mole) reagent designed to

acylate amino groups has been synthesized. This compound, the sulphone of
35S-labelled formyhnethionyl methyl phosphate, cannot pass through the red
blood cell membrane. When added to intact red blood cells, it reacts with, and
labels, two proteins on the outside surface of the cell membrane: these have mole-
cular weights of about 106,000 and 90,000 daltons. All membrane proteins appear
to be labelled if erythrocyte ghosts are exposed to the reagent. The two proteins
exposed on the outside surface of the erythrocyte are major membrane
components: they probably cover a significant portion of the surface of the cell.
The smaller of these two proteins carries the bulk of the cell surface sialic acid
and a large portion of cell surface carbohydrate. The other, a larger protein, carries
very little, if any, carbohydrate or sialic acid.

1. Introduction
Cell membranes of human erythrocytes contain approximately equal amounts of
protein and lipid by weight; the carbohydrate content is comparatively small. Many
different proteins, with a wide range of molecular weights, are found associated with
these membranes (Rosenberg & Guidotti, 1968,1969).Most of the sialic acid present
in the erythrocyte is bound, through other carbohydrates, to proteins on the outside
surface of the cell (Cook $ Eylar, 1965).
In order to find out how these proteins are arranged in the lipid bilayer, I
have prepared a reactive and radioactive reagent of very high specific activity
which appears to be unable to pass through the cell membrane. This reagent, the
[35S]sulphone of formylmethionyl methyl phosphate,

-O--+8 =o
I
C&a
I
CJ% 0
II
OCH-NH-CR-CO-0-P-0-CHs
I
-0
Formylmethionyl &phone methyl phosphate
776
776 M. S. BRETSCHER
labels all the proteins of the red blood cell ghost (in which both sides of the membrane
are exposed). However, only two of the major protein components are labelled if
intact cells are treated with FMNPt. The smaller of these two “outside” proteins
appears to carry a large portion of the cell surface sialic acid and carbohydrate.
Whilst this work was in progress, several other groups have published results which
are in general agreement with the picture presented here (Berg, 1969 ; Lenard, 1970 ;
Phillips & Morrison, 1970; Bender, Garan & Berg, 1971).

2. Materials and Methods

(a) Fownylmethionyl &phone methyl phoephale
(i) 6’yntheai.s
[36S]Methionine of very high specific activity (20 Ci/m-mole) was prepared essentially as
described by Sanger, Bretscher & Hocquard (1964), except that Escherichiu coli was
grown on a minimal sulphate-free medium in the presence of 1 mCi/ml. [36S]sulphate in
0.05 maa-sulphate. Alternatively, it was purchased from the Radiochemical Centre,
Amersham, England. The methionine was converted to its N-formylated derivative by
reaction with formyl-N-hydroxysuccinimide and was then oxidized to the sulphone.
Crude formyl-N-hydroxysuccinimide was prepared as follows: 50 ml. 98% formic acid
and 18 ml. acetic anhydride were mixed at room temperature for 16 min and then chilled to
0°C. To this were added 6 g solid N-hydroxysuccinimide: the mixture was stirred for 2 hr
at 0°C and then for 1 hr at about 22°C. The whole was then dried on a rotary evaporator,
care being taken to exclude all moisture. The solid residue was crushed and the remaining
acetic and formic acids removed in wucuo over solid NaOH pellets and concentrated
H2S04. Conversion of methionine to formyhnethionine was achieved by dissolving the
methionine in 0.1 ml. water and adding to this 0.06 ml. dioxane and 0.01 ml. triethylamine;
this was chilled to 0°C and 0.1 ml. of a solution of 100 mg crude formyl N-hydroxysuccini-
mide in 1 ml. dioxane added. The mixture was immediately vortexed and held at O’C for
5 min or longer. Conversion of methionine to its formylated derivative is quantitative;
the product was isolated by electrophoresis at pH 35 (60 v/cm for 1 hr).
Formyhnethionine was eluted with water and a solution of 100 pg of carrier bovine serum
albumin added. The whole was mixed and dried down C VUCUO.Oxidation with O-2 ml.
performic acid (prepared from 0.96 ml. of 98% formic acid and 0.06 ml. of 30% hydrogen
peroxide) was carried out at OW for 2 hr. The formic acid was removed in vacua and the
sulphone of formylmethionine isolated again by electrophoresis at pH 3.6 (1 hr at 60 v/cm).
Formylmethionyl sulphone methyl phosphate was prepared by a modification of Berg’s
procedure (1968). Formylmethionine sulphone (1 to 6 mCi) was dried down in a small test
tube (capacity 0.6 ml.). It was dissolved in 6 d. water and 10 d. pyridine, to which were
added 2 ~1. 1 M-sodium methyl phosphate (Koch-Light Labs, England) and 1 d. 12 N-HCl;
this gives a clear solution. To this were added 26 d. of a solution of dicyclohexylcarbodiii-
mide in pyridine ( 1 g dicyclohexylcarbodiimide + 1 ml. pyridine) and the reaction vortexed
and chilled to O’C. Sticient pyridine (about 40 ~1.) was then added to maintain a single
phase at 0°C. The tube was held at 0% for 3 hr, and vortexed once each hour. Solid dkyclo-
hexylurea precipitates during the reaction.

(ii) Isolation
The contents of the reaction tube were transferred to a small piece of 3 MM chromato-
graphy paper (2 cm x 3 cm shaped with a tip) and quickly dried at room temperature. The
paper wea immediately washed twice in 100 ml. ether to remove dicyclohexylcarbodiimide;
FMMP was then eluted at 4W with 10e3 N-HCl. About 60 $. eluete were collected, frozen
and stored in liquid nitrogen.
The over-all recovery of radioactivity (starting from methionine) was approximately

t Abbrevimtions used: FMMP, formyhnethionyl sulphone methyl phosphate; dodecyl SO,,

sodium dodeoyl sulphate.
 ERYTHROCYTE MEMBRANE PROTEINS 777
50 to 80%. The FMMP was assayed by its reaction with 0.1 M-glycine in sodium carbonate
bicarbonate buffer (O-06 M in each) to give formyhnethionyl sulphone glycine ; hydrolysis by
buffer alone yields only formylmethionine sulphone. Both these products were detected by
paper electrophoresis as described previously (Bretscher, 1969). About 60 to 9O”/e of the
label present in the above preparation reacted to give formylmethionyl sulphone glycine.
FMMP thus prepared still contains unlabelled methyl phosphate, from which it can be
separated by electrophoresis at pH 3.5. For the present experiments this was not found to
be necessary.
(b) Red blood cells and ghosts
Human blood (A, Rh positive), no older than 10 days, was used. The red cells were
washed 4 times in 0.9% NaCl to remove the buffy coat and serum proteins. Ghosts were
prepared from them by lysis in at least 10 vol. of 0.01 M-sodium phosphate (pH 7.2)
containing 10v5 M-&C&. These were isolated by centrifugation and washed 5 times in t,he
lysis buffer. The final colour of different preparations ranged from pink to white. Intact
cells or ghosts were used immediately.
(i) Labelling procedure
Before labelling, cells or ghosts were washed once in a carbonate-bicarbonate buffer
solution containing 0.05 M-NasCOs, 0.05 M-NaHCO, and 0.05 M-N&~, pH 10. Red cells are
surprisingly stable under these conditions. 0.2-ml. packed red cells or O-2-ml. of a ghost
suspension (derived from about 0*2-ml. packed cells) was placed in a test tube wit,h 0.1 ml.
carbonate-bicarbonate buffer at 0°C. 35S-labelled FMMP (1 to 10 ~1.) was added, the
suspension immediately vortexed and held at room temperature for 10 min. The suspension
was then diluted with either 0.90/, N&l (for red cells) or 0.01 M-sodium phosphate, pH 7.2,
containing 10e5 M-C&l, (for ghosts), and washed with 10 ml. buffer at least 4 times, The
extent of lysis of whole cells during the labelling procedure does not exceed 5% (and is
usually less than half this value) as measured by the release of haemoglobin into the cell
supernatant.
(ii) Polyacrylamide gel analysis
Ghosts were dissolved in a solution of dodecyl SO,, so that at least 5 mg dodecyl SO,
were present per mg protein. To inhibit proteases, phenylmethane sulphonyl fluoride
dissolved in n-propanol was added to a final concentration of 2 mM and j?-mercaptoethanol
to 2%; the sample was heated to 60°C for 5 min. Ghost solutions in dodecyl SO4 were
always stored at - 20°C and were not usually dialysed.
Acrylamide gels (7.5%, 1.1 cm in diameter, 12 cm long) were run under standard con-
ditions in O*l% dodecyl SO*, 0.1 M-sodium phosphate, pH 7.2, at room temperature
(Shapiro, Vifiuela & Maizel, 1967). The ghost sample, containing about 100 pg membrane
protein, was diluted to 0.5 ml. so that the final concentrations in the sample were
2% dodecyl SO,, 0.01 M-sodium phosphate (pH 7~2)~ 0.5% /%mercaptoethanol, 0.4
mw-phenylmethane sulphonyl fluoride, 0.02% bromphenol blue (dye marker) and
10% glycerol. Ghost samples in which sialic acid was to be monitored along the gel were
prepared in the same way but contained 4 M-urea in place of the 10% glycerol.
In all cases the gel was sliced longitudinally and a section 2.5 mm thick removed. This
was usually stained either with Coomassie brilliant blue for proteins (Weber & Osborn,
1969), or with the periodate-Schiff stain for carbohydrates (Zacharius, Zell, Morrison 8:
Woodlock, 1969). For autoradiography, the middle section of a gel containing between
5 x lo4 and 5 x lo5 cts/min w&8 dried on filter paper and exposed to X-ray film (Laemmli,
1970). Molecular weights were estimated by comparison with markers of RNA polymerase
(165,000, 160,000, 90,000 and 40,000 subunits) and ribonuclease (13,500).
(iii) Sialic acid assay
The Warren (1959) assay was used, modified to sccommodete smaller volumes. The
lyophilized sample was heated in 0.2 ml. of O-1 N-H~SO~ for 1 hr at 75°C to liberate the
sialic acid. 0.03 ml. of 0.07 M-sodium periodate in 9 M-phosphoric acid was added and
the sample held at room temperature for 20 min. 0.1 ml. of 10% sodium arsenite in 0.5
M-Na,SO, and 0.1 M-H$SO~ wa,s added, the sample was vortexed and placed on a mechanical
 778 M. S. BRETSCHER
shaker and shaken vigorously at room temperature for 30 min. O-06 ml. of 6% sodium
thiobarbiturate was added and the sample heated in a boiling water bath for 10 min.
Cyclohexanone (0.6 ml.) was added, the sample vortexed, chilled to below room tempera-
ture, vortexed again and finally centrifuged to separate clearly the two phases. Of the
upper organic phase, 0.5 ml. was removed and its optical density at 549 nm measured. The
total absorbancy at 549 nm produced in this modified assay is the same as that produced,
per pg sialic acid, in Warren’s (1959) method.
When gel slices were assayed for sialic acid, a gel slice (about O-1 ml. in volume) was
soaked in 1 ml. 0.06 N-HaSO, for 2 hr, heated for 1 hr at 75°C and then shaken overnight at
room temperature. The supernatant solution was concentrated to 0.2 ml. in vucuo and then
assayed as above.

3. Results
(a) Irnpemneubility of red blood cells to formylmethionyl sulphone methyl phosphate
Washed red blood cells were labelled with FMMP and then washed to remove any
remaining reagent. They were lysed and their membranes isolated by centrifugation:
the red supernatant was saved. The membranes were further washed at low ionic
strength. In Table 1 are shown the specific activities of the ghost and haemoglobin

TABLE 1
flpecific activities of haemoglobin and membrane proteins after lubelling
with formylmethionyl sulphone methyl phosphate

Preparation Protein Radioactivity Specific aotivity

Fraction
labelled h3) (cts/min) (cts/min/mg)

1. Red cells Ghosts 0.6 6~10~ 106

Hb 45 1.1 x 106 2.5 x 103
2. Haemoglobin Bib 25 5.45x 10’ 2.2 x 106

Preperation 1. 0.2~ml. loosely packed red cells were labelled with about 3 x lOa cts/min of
FMMP as described in Materials and Methods.
After washing, the cells were lysed and the ghost and haemoglobin fractions collected, dissolved
in dodecyl SO, and dialysed against 0.1% dodecyl SO1 in 0.01 M-sodium phosphate pH 7.2. Protein
was determined as described by Lowry, Rosebrough, Farr & Randall (1951); radioactivity was
measured aa perchlorio acid-precipitable radioactive material.
Preparation 2. Haemoglobin was labelled under the same conditions as in preparation 1 with
3 x lo* cts/min FMMP and then dialysed against 0.02 M-Tris*HCI, pH 7.4. As can be seen, about
20% of the label in the added FMMP became covalently bound to the haemoglobin.

fractions, measured as the acid-precipitable radioactivity per milligram protein. The

haemoglobin is very poorly labelled : its specific activity is 400 times lower than the ghost
proteins. In a parallel experiment, (preparation 2, Table I), free haemoglobin was found
to be heavily labelled by FMMP. These results show that FMMP cannot readily react
with components enclosed by the cell membrane; they suggest that the membrane is
reasonably impermeable to the reagent.
(b) Which proteins are lube&d?
Ghosts and whole cells were labelled with FMNP. The membranes from each pre-
paration were dissolved in dodecyl SO, and analysed on polyacrylamide gels. Plate I
shows that there are many bands which stain with Coomassie brilliant blue in these
preparations: they are presumably proteins, (gels Al and A2).
 Hb

I Dodecyl , SO4 front

Al

BI

I’LATE I. Gel electrophoresis of membranes (A) and its autoradiogram (R) of mwnhrancs f’unn
labclid ghosts (1) and Isbelled red blood cells (2).
Samples for electrophoresis mere prepared and run as deacribcd in hlatcrials and lilrthotis.
Elvct~rophoresis was performed for 8 hr at, 2 V/cm at room temperature. The middlr sect ioll of thl~
,gvI 2.5 mm thick was stained and photographed (for -4) ant1 then antol,atliograph(~tl four 21 hr
(flu, R).
 Origin a b
Dodecyl SO, front

I ii +

I IO 20 30 40 50 60 70 80
Fraction no

I'LATE II. Sialic acid content of membrane pmteirls

4 sample of membranes (about, 150 pg of protein containing 12 pg sialic acid) was dissolvctl in
detergent and run on a gel as described in Materials and Methods. The go1 was sliced to remove a
2.5.mm thick longitudinal section from the middle: this was stained for protein and is shown
above. The remainder of the gel was sliced into 84 fractions, each of which was assayed for sialic
acid. The recovery of sialic acid (including that, in the stained section) was about 70%. Prior
t,reatment of whole cells with neuraminidase (0.2 ml. packed cells in 0.4 ml. of 0.15 M-NaCl,
0.05 &I-sodium phosphate (pH 6.0) and 5 pg Sigma neuraminidaRe (2 units/mg) for 1 hr at 37”C,
conditions which removed about SOY/, of the sialic acid from the cell membranes) removes all the
detectable sialic acid from fract)ions 1 I to 28. In other experiments relatively more sialic acid was
found in the region around fraction 54.

PLATE III. Gel stained with the Schiff stain for carbohydrate
This gel was run as that in Plat,e II. The gel was stained for carbohydrate as described by
Zacharius et al. (1969) and photographed (i); this same slice was then stained with Coomassie
brilliant blue, (ii).
 ERYTHROCYTEMEMBRANEPROTEINS 779

When intact erythrocytes are labelled with F’MMP, only two protein componenm
are heavily labelled (Plate I, B2). These have apparent molecular weights of about
105,000 (band a) and 90,000 (band b) respectively. Under similar conditions, treatment
of ghosts with FMMP labels all the visible protein bands (Plate I, Bl). These results
suggest that the proteins in bands a and b are partly located on the external surface
of the red blood cell. The autoradiograms in Plate I also show that some small com-
ponents which migrate with the dodecyl SO, front on these gels become labelled: the
nature of this material is uncertain.

(c) Which proteins cary sialic acid and carbohydrates?

The bulk of the red blood cell sialic acid is known to be located on the external
surface, joined to protein (Cook & Eylar, 1965). Plate II shows the middle section of a
dodecyl SO, gel of ghost proteins stained with Coomassie brilliant blue. The two side
portions of the same gel were cut into 84 segments and each assayed for sialic acid
(Plate II). This reveals that most of the sialic acid present in the ghost migrates on the
gel together with band b : the sialic acid is presumably covalently joined to band 6.
There are two other regions in the gel which contain lesser amounts of sialio acid:
some is found near the dodecyl SO, front. The latter, in different experiments, ac-
counts for between 15 to 30% of the total sialic acid. Treatment of whole cells with
neuraminidase under conditions which removes about 80% of the total red cell
ghost’s sialic acid content quantitatively removes the sialic acid from band b. The
sialic acid migrating in the dodecyl SO, front of the gel is not so readily removed.
Plate III shows another dodecyl SO, gel of membrane proteins which has been first
stained for carbohydrate as described by Zacharius et al. (1969) and subsequent,ly
stained for protein. Band b appears to carry a large portion of the cell surface carbo-
hydrate; most of the remainder migrates with the dodecyl SO, front. The carbo-
hydrate stain thus reveals a picture similar to that seen with the sialic acid assay.

4. Discussion
The membranes of red blood cells are known to contain a variety of proteins
(Rosenberg & Guidotti, 1968). These can be fractionated according to their molecular
weights in dodecyl S0,-7.5°/o polyacrylamide gels: under favourable conditions at
least twenty distinct bands can be seen. The largest major components are not well
resolved here : however, on 5% acrylamide gels two proteins, with molecular weights
of around 200,000, are clearly seen. The next major components, band a (105,000
daltons) and band b (90,000 daltons) are of particular interest and will be discussed
below. There are six other prominent bands with molecular weights between about
80,000 and 12,000 daltons. All these different components have been observed by
several other groups (Berg, 1969 ; Lenard, 1970 ; Phillips & Morrison, 1970 ; Bender
et al., 1971). Whether all these proteins are associated with the membrane in the cell
we do not know-there is no clear criterion by which to judge which are membrane
proteins and which are just “sticky”.
When intact red blood cells are labelled with FMMP, only the proteins in bands a
and b are readily labelled. In contrast, all proteins are labelled if erythrocyte ghosts
are exposed to FMMP. This shows that these proteins in bands a and b reside, at least
in part, on the outside surface of the cell: they are almost certainly real membrane
components. What is surprising is that one of them (band b) carries most of the cell
780 M. 5. BRETSCHER

surface sialic acid and carbohydrate. Band a appears devoid of either. Since the protein
in band b is smaller than that in a, and in addition carries much carbohydrate, the
proteins in these two bands must be different. I estimate, by the intensity with which
bands a and b are stained, that each represents about 15% of the membrane protein
(excluding contaminating haemoglobin). Taking Rosenberg & Guidotti’s (1968) data
for the amount of sialic acid present in haemoglobin-free ghosts, I estimate that there
are at least 40 residues of sialic acid per protein molecule in band b (assuming only
one kind of polypeptide chain in band b).
That the proteins in bands a and b are not trivial components of the outside surface
of the erythrocyte is clear from the large amount of each present. 1 ml. of packed red
cells contains about lOlo cells (see the review by van Deenen BEde Gier (1964) for many
useful parameters of red cells) with a surface area on each of about 12 x lOlo A”.
lOlo cells contain about 5 mg of membrane protein, from which one can calculate that
each cell must contain approximately 5 x lo5 copies of the proteins in bands a and b
(again assuming these to be single species). Furthermore, if their dimensions are taken
to be similar to hexokinase (a globular protein of about 100,000 daltons, whose dimen-
sions are 50 A x 50 A x 100 A approximately, T. A. Steitz, personal communication),
each molecule would have l-2 x lo4 A” if these proteins are evenly distributed over the
cell surface. If the long axis of each protein is placed perpendicular to the membrane
surface, we find that about 20% of the outer surface of the membrane is covered by
the protein in both bands a and b. So far, attempts to fingerprint these proteins (when
labelled from the outside of the cell only, and when labelled from both sides) have
been unsuccessful. It would clearly be of interest to know whether they pass from the
outside to the inside surface of the cell membrane.
The autoradiograms, sialic acid assay and carbohydrate stain all show some material
which migrates with the dodecyl SO, front which is not stained with Coomassie
brilliant blue. The nature of this material is uncertain (although it may contain small
protein molecules (Laico, Ruoslahti, Papermaster & Dreyer, 1970)).
The reagent used here, 35S-labelled formylmethionyl sulphone methyl phosphate,
has been specifically designed to label the surfaces of cells : the negative charge and the
presence of the sulphone residue probably prevent it from passing through the lipid
bilayer. It is a powerful acylating reagent which would be expected to add a formyl-
methionyl sulphone residue onto amino and possibly hydroxyl groups. In separate
experiments I found that most of the label transferred onto haemoglobin at pH 10 is
in fact attached to the c-amino group of lysine. The extremely high specific activity of
FMMP (10 to 20 Ci/m-mole) makes it useful for labelling proteins which are either
present in very small amounts or which can only be fractionated on a microgram
scale. More important for the work reported here, its high specific activity enables
sufllcient label to be incorporated with only slight chemical modification of the
structure under study. A reagent of lower specific activity might require the saturation
of reactive groups with possible distortion of the membrane during the labelling pro-
cedure. The presence of the formyl group, which can later be selectively removed from
a formylmethionine peptide, could be used for diagonal purification procedures.

I am most grateful to Dr Beverley GrifXn for much chemical advice. I thank Drs Mary
Osborn, Joan Argetsinger Steitz, U. Laemmli and J. Maize1 for numerous helpful discus-
sions and Mr W. W. Bender for showing me his results before publication. I also thank
Mrs M. S. Brownlee for technical assistance and the Regional Blood Transfusion Service of
Addenbrooke’s Hospital for the supply of blood.
 ERYTHROCYTE MEMBRANE PROTEINS 781

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