THE JOURNAL OF BIOLOGIC.~L CHEMISTRY Vol. 243, No. 8, Issue of April 25, pp. 1935-1992, 1968

Printed in U.S.A.

The Protein of Human Erythrocyte Membranes

I. PREPARATION, SOLUBILIZATION, AND PARTIAL CHARACTERIZATION*

(Received for publication, October 16, 1967)

STEVEN A. ROSEXBERG$. AND GUIDO GUIDOTTI

From the Biological Laboratories, Harvard University, Cambridge, Massachusetts &?I$8

SUMMARY

This paper presents studies on the characterization of the protein component of mammalian erythrocyte membranes. Exhaustive lipid extraction of the membrane results in a glycoprotein residue containing greater than 90% of the total membrane protein. The composition of this membrane preparation is presented. Several methods have been developed to solubilize this protein. The protein is soluble in formic acid and sodium dodecyl sulfate solutions. After total succinylation of the protein, complete solubility is achieved in aqueous solutions at neutral pH. A new method for completely solubilizing the unmodified membrane protein in urea and guanidine hydrochloride solutions at neutral pH in the absence of detergent is presented. This method de- pends on initial solubilization of the membrane protein in phenol. End group analysis, electrophoresis, ultracentrifu- gation, and chromatography in these solvent systems reveal the protein in membranes to be a heterogeneous collection of proteins, many in the molecular weight range near 50,000. The membrane preparation and the solvent systems described will be valuable in the characterization of the individual protein molecules present in the membrane. These studies are now in progress.

All known plasma membranes are composed primarily of lipid and protein. The lipid components of membranes have been extensively studied (l-4). The nature of the protein found in membranes is not well understood, however. Little is known concerning the number or size of membrane proteins, the com- position of individual proteins, the nature of the protein-lipid interaction, or the identity and role of those proteins important in membrane transport.

A major difficulty in the study of membrane proteins arises

* This work was supported by United States Public Health Service Grant 08893 and by National Science Foundation Grant GB-6360.

# Postdoctoral Fellow in the Department of Biophysics, Har- vard University, supported by United States Public Health Service Grant l-F3-GM-32,453-01.

from the ability of these proteins to bind lipid tenaciously and form large nonspecific aggregates in aqueous solution. Partial lipid extraction of membranes, however, results in a very in- soluble residue.

We have undertaken a study of the chemical nature and function of the protein component of mammalian cell membranes. Human erythrocytes were used as the source of membranes because of the relative ease of obtaining membrane preparations uncontaminated by intracellular organelles. Our approach has utilized a technique for totally extracting the lipid from mem- brane preparations, thus circumventing problems of protein aggregation with lipid. Several techniques for solubilizing the glycoprotein residue have been developed. This paper deals with the techniques for the preparation and solubilization of the lipid-free membrane protein. The composition, number, and size of the membrane proteins are discussed.

Further studies are in progress dealing with the separation and analysis of the individual protein components as well as attempts to identify those proteins important in the antigenic and transport functions of the cell.

MATERIALS AND METHODS

Chemicals

All chemicals used were reagent grade unless otherwise speci- fied. Water used in all solutions was distilled and deionized. Phenol was distilled and stored at 4”.

Immediately before use, 8 M urea solutions were stirred with a mixed bed ion exchange resin (Bio-Rad AG 501-X8) and acti- vated charcoal for several hours and then filtered through Celite. Early experiments were done with urea recrystallized from 70% alcohol.

Guanidine hydrochloride was prepared from guanidine carbonate by the method of Anson (5). Sodium dodecyl sulfate was recrystallized from 80% ethanol and stored as a 10% stock solution at room temperature. Thiobarbituric acid used in the assay for sialic acid was recrystallized from boiling water

(6).

Assays

Amino Acid AnalysisThe protein (see below) was hydrolyzed in evacuated sealed tubes in 6 N HCl at 110’ for varying times

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Protein of Human Erythrocyte Membranes. I Vol. 243, No. 8

up to 72 hours. The solution was evaporated to dryness and the residue was suspended in 0.2 N citrate buffer, pH 2.2. Anal- yses were performed on a Spinco model 120C automatic amino acid analyzer with the system of Moore and Stein (7). Separate samples were oxidized with performic acid and assayed for cysteic acid (8). Tryptophan was estimated spectrophoto- metrically by the method of Beaven and Holiday (9) after the protein had been heated in 0.1 N NaOH for 70 min.

Protein-The procedure of Lowry et al. (10) was used. Bovine serum albumin (Mann, Fraction V) was used as a standard. The ninhydrin assay of Moore and Stem (11) was used for the determination of free ammo groups. In the assay for protein of fractions from sucrose gradients or chromatographic columns, alkaline hydrolysis of an aliquot at 110” for 2 hours preceded the ninhydrin assay.

Lipid-Total phosphorus was measured by the method of Bartlett (12). In the absence of other phosphorus-containing molecules the amount of phospholipid could be calculated from the amount of phosphorus present. Cholest.erol was assayed by the method of Zlatkis, Zak, and Boyle (13).

Carbohydrate-Neutral sugars were assayed by the method of Dubois (14) with galactose as a standard. Sialic acid was measured by the thiobarbituric acid assay described by Warren (6).

Glucosamine and galactosamine were assayed by the technique of Walborg (15) with the Spinco automatic analyzer with a 50-cm column equilibrated with 0.2 N citrate buffer at pH 5.28. Gluco- samine and galactosamine from Mann were used as standards. Since this method detects only free amino sugars, hydrolysis of the protein was necessary prior to the sugar assay. To de- termine the optimal hydrolysis time, i.e. maximum release with minimum destruction of the sugars, identical samples were hydrolyzed for varying times in 3 N HCl. As can be seen in Fig. 1, optimal release of glucosamine and galactosamine occurs at about 19 hours in 3 N HCl at 110”. This time was used in all analyses. No correction was made for destruction of the sugars.

Hemoglobin-The pyridine hemochromogen method as de- scribed by Dodge, Mitchell, and Hanahan (16) was used. This technique is sensitive to concentrations of hemin down to 2 x low6 g per 100 ml.

Carbonic Anhydrme-Carbonic anhydrase activity was assayed by the calorimetric method of Wilbur and Anderson (17). The assay was performed on freshly prepared ghosts (see below)

20 30 40 50 60 HYDROLYSIS TIME (HOURS)

FIG. 1. Hexosamine recovery after hydrolysis of erythrocyte membranes for varying times in 3 N HCl at 110’. Glucosamine and galactosamine were determined chromatographically (15).

in both 0.005 M Tris hydrochloride and 0.005 M phosphate buffer at a pH of 7.5.

NH&rminal Amino Acids--The cyanate method of Stark and Smyth (18) was used. The initial reaction of the membrane protein with cyanate was performed in both 1% SDS and in 6 M guanidine hydrochloride. Similar results were obtained in the two solvent systems. Analysis was performed only for the neutral and acidic amino acids excluding cysteine and tryptophan.

Ultracentrifugation Studies

All sedimentation velocity and equilibrium experiments were performed at 20’ in a Spinco model E ultracentrifuge equipped with both schlieren and Rayleigh interference optical systems. Photographic plates were measured on a Gaertner microcom- parator.

Sedimentation velocity experiments were run at 59,780 rpm in standard double sector centerpieces. Experiments in high formic acid concentrations were performed in a Kel-F single sector centerpiece. A capillary type interference centerpiece was used for synthetic boundary runs in guanidine hydrochloride solutions. Sedimentation coefficients were converted to szo,ut values with corrections from the International Critical Tables (19). Corrections were computed from the data of Kawahara and Tanford (20) when guanidine hydrochloride solutions were used.

Sedimentation equilibrium experiments were performed by the meniscus depletion method of Yphantis (21) in 12-mm triple channel centerpieces. Operating speeds were between 25,000 and 40,000 rpm and at least 20 hours were allowed for attaining equilibrium. Solution densities in these experiments and those for measuring the partial specific volume of the protein (see below) were measured in lo-ml Gay-Lussac pycnometers. Calculation of the weight average molecular weight (#,J was performed as detailed by Yphantis (21).

The partial specific volume of the protein (v) was measured by comparing the sedimentation of the ghost protein in 1% SDS solutions prepared with either Hz0 or D,O by a method sug- gested by Edelstein and Schachman (22). These solutions were run simultaneously with two of the three channels of a 12-mm Yphantis cell. v was also calculated from the amino acid composition by the method of Cohn and Edsall (23) and cor- rected for the v of the measured carbohydrate present with the data of Weber (24) and Popenoe and Drew (25).

Sucrose density gradient centrifugation was performed at 4” in 5-ml cellulose nitrate tubes in a swinging bucket 50 rotor with a Spinco model L ultracentrifuge at, 50,000 rpm for 6+ hours. Gradients were from 20 to 5% sucrose. The approximate sizes of components were estimated from the sedimentation of 4 S and 16 S ribonucleic acid under similar conditions.

Column Chromatography

The membrane protein in 1% SDS was chromatographed on gel filtration medium Sepharose 4B (Pharmacia). The columns, 2.5 x 190 cm, were packed and equilibrated with several column volumes of 1% SDS solution. The flow rate of the column was 20 ml per hour. Fractions (4 ml) were collected and moni- tored by ninhydrin assay after alkaline hydrolysis. Bovine serum albumin and lysozyme were chromatographed on the

* The abbreviation used is: SDS, sodium dodeeyl sulfate.

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same column under identical conditions to serve as molecular weight markers. Different amounts of bound SDS add to the error of this column calibration.

Succinylalion

Succinylation of the membrane protein was performed at room temperature by a method adapted from Hass (26). Solid succinic anhydride was added with stirring to a 1% solution of membrane protein in 8 M urea solution. About lOOO-fold molar excess of succinic anhgdride was used. The pH of the reaction was maintained between 8 and 9 by the gradual addition of NaOH either automatically, with a Radiometer Titrigraph, or manually. The reaction mixture was dialyzed against the desired buffer, concentrated against dry Aquacide (Bio-Rad), and dialyzed again. The completeness of succinylation of amino groups was tested by comparing protein (method of Lowry et al. (10)) and amino group (ninhydrin reaction) assays on aliquots of the protein solution before and after succinylation. In this way a measure of the number of free amino groups for a known amount of protein was obtained.

Polyacrylamiok Gel Electrophores-is

Several electrophoretic systems were utilized. At pH 8.7 the general method of Ornstein (27) and Davis (28) was used with and without 8 M urea in both the spacer and separating gels. Acrylamide concentrations from 4 to 7.5% were used in the separating gel compartment. The sample was added in 1% SDS and electrophoresis was at room temperature.

Gel systems containing 0.1% SDS were prepared by the method of Vifiuela, Algranati, and Ochoa (29). All gels in SDS contained 10% acrylamide. Staining of the gels was performed with 1% Amido-Schwartz in 7.5% acetic acid or 0.25% Coomassie brilliant blue in methanol-acetic acid-water (5: 1:5).

Preparation of Lipid-extra&d Membrane Protein

Human blood, no more than 1 week old, was used. All blood was type 0, Rh-positive. Packed cells from up to five donors were processed at a single time. The entire procedure was performed at 4” (see Fig. 2).

The packed cells were centrifuged for 20 min at 5000 X g and the plasma and b&y coat were aspirated. The cells were then suspended in an equal volume of 310 mOsM Tris-HCl buffer at pH 7.5 and washed three times. Red cells were freely sacrificed in all wash steps to ensure complete removal of the b&y coat. The supernatant was clear and colorless after the final wash.

Hemolyzed erythrocytes (ghosts) were prepared by the method of Dodge et a2. (16). The packed red blood cells were suspended in a 30-fold volume of 15 mOsM Tris-HCl buffer at pH 7.5, mixed, and allowed to stand for 1 hour. For the prep- aration of small amounts of membrane protein centrifugation was then performed in 200-ml polypropylene bottles at 13,000 X g for 40 min in a Sorvall RC-2 centrifuge. For preparations of large amounts of protein, a Szent-Gyorgi and Blum continuous flow apparatus (Sorvall) was used at 20,000 X g and the ghosts were collected in 50-ml stainless steel cups.

The residue was resuspended in another 30-volume of the same buffer and the wash was repeated three times. After the final wash the ghosts were creamy white with no trace of redness.

The technique of lipid extraction was similar to that used by Scanu, Lewis, and Bumpus (30). The pellet of hemolyzed

Packed red blood cells

1 I

Centrifuge

Supernatant Red blood cells Plasma Buffy coat Wash with 310 mOsM buffer

1 I

Centrifuge

Supernatant Washed red blood cells Plasma Buffy coat

1~

I

Hemolyxe in 15 mOsM buffer Centrifuge (three times)

Supernatant Washed red blood cell ghosts Hemoglobin Other intracellular

material

I

Extract with ethanol-ether (3:l)

r------ Centrifuge Lipid Partially lipid-extracted ghosts

71 Extract with ether in modified Soxhlet

apparatus for 48 hours Lipid Lipid-free membrane protein

JDry Final membrane protein powder

FIG. 2. Preparation of lipid-extracted membrane protein

erythrocytes was mixed with a 15-fold volume of ethanol-ether (3:l) at -15”. After 2 hours the precipitate was collected by centrifugation for 30 min at 13,000 x g. The precipitate was resuspended in ethanol-ether as above and the above extraction was repeated two more times.

The residue was collected by filtration through Whatman No. 52 filter paper after the final extraction. The residue was wrapped in the filter paper and put in a Soxhlet apparatus which was modified to allow continuous ether extraction while main- taining the protein temperature below 0”. With this apparatus the protein was ether-extracted every 5 to 10 min for 48 hours. The temperature of the bath was kept at -70” with Dry Ice and acetone. Some experiments were performed on protein extracted at room temperature. No differences were noted between the protein prepared at the two different temperatures. The residue was then dried over Drierite in a desiccator for 24 hours and stored at 4”.

About 290 mg of dried protein powder were obtained from each 300 ml of packed red cells.

RESULTS

Composition of Lipid-extracted Membrane Protein-Assay of the red blood cell ghosts revealed that less than 0.4% of the total remaining protein was hemoglobin. Less than 0.01% of the remaining protein was carbonic anhydrase.

The supernatant solutions from the lipid extractions were concentrated in a rotary evaporator and assayed for protein. An average of 6.5% of the total protein was extracted by alcohol- ether and an additional 2.2% by the ether extraction. Assay of the membrane protein powder revealed from 1 to 2% phospho- lipid (as measured by total phosphorus) and no cholesterol. The final protein powder thus contains about 91% of the total membrane protein. The extracted protein may correspond to that tightly bound to lipid.

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1988 Protein of Human Erythrocyte Membranes. I Vol. 243, No. 8

The compositions of the intact and lipid-extracted ghosts are given in Table I. The ammo acid composition of the lipid- extracted membrane protein is given in Table II.

From the total measured composition of the membrane protein powder the expected elemental composition may be calculated.

TABLE I Composition of intact and lipid-extracted red blood cell ghosts

component Intact ghost Lipid-extracted ghost

% %

Protein ....................... 49.2 91 .o Lipid (total) ................. 43.6 1.5

Phospholipid ............... 32.5 1.5 Cholesterol . 11.1 0

Carbohydrate (total) ......... 7.2 7.5 Neutral sugars. ............ 4.0 2.5 Hexosamines ............... 2.0 2.6 Sialic acids. ............... 1.2 2.4

TABLE II Amino acid composition of lipid-extracted membrane protein

Amino acid Residues per 100 residues

Lysine ................................ Histidine ............................. Arginine .............................. Aspartic acid”. ........................ Threonine. ........................... Serine ................................ Glutamic acid”. ....................... Proline ............................... Glycine ............................... Alanine ............................... Half -cystine .......................... Valine ................................ Methionine ........................... Isoleucine ............................ Leucine .............................. Tyrosine ............................. Phenylalanine ........................ Tryptophan ...........................

5.21 2.44 4.53 8.49 5.86 6.26

12.15 4.26 6.73 8.15 1.08 7.10 2.02 5.29

11.34 2.41 4.20 2.49

0 One mole of NH3 was formed for every three azpartic and glutamic acids.

TABLE III Elemental analysis of lipid-extracted membrane

Element Expected percentage Measuredpercentage”~*

Carbon .................... Nitrogen. .................. Hydrogen. ................. Phosphorus. ............... Oxygen. .................... Sulfur ......................

45.26 49.83 13.50 14.49

7.60 7.40 0.05 0.06

33.09 27.57~ 0.65 0.73d

a Performed by Dr. S. M. Nagy, Department of Chemistry, Massachusetts Institute of Technology, Boston, Massachusetts.

* Average of two measurements (fl%). 0 Not measured; obtained by difference. d One measurement.

All of the phospholipid was assumed to be phosphatidyl choline and all fatty acid side chains were assumed to be oleic acid. Although other lipids are present in the membrane in equiv- alent amounts, these assumptions serve to simplify the approxi- mation and are valid within the error of the approximation. The expected and measured results of the elemental analysis are given in Table III.

Solubility of Lipid-extracted Membrane Protein-Attempts were made to solubiliae the membrane protein powder in a wide variety of solvents by mixing 1 to 2 mg of the protein with 2 ml of the solvent and shaking vigorously for 12 hours. Protein that did not sediment when centrifuged at 50,009 x g for 1 hour was considered to be soluble.

The protein was insoluble in formate, acetate, phosphate, Tris, and borate buffers in the pH range from 3 to 10. Only a small fraction of the powder dissolved in 8 M urea, 8 M guanidine hydrochloride, 1 M MgC&, dimethyl sulfoxide, hexafluoroacetone, glacial acetic acid, and Triton X-100 solut.ions with and with- out 8 M urea.

More than 97% of the powder is soluble in 88% formic acid and, although the protein cannot be solubilised by 0.9% formic acid, it did not precipitate when dialyzed into this solvent from 88% formic acid. The protein precipitates when dialyzed into 8 M urea at neutral pH.

More than 98% of the membrane protein can be dissolved by shaking in 1% SDS. Lower concentrations of SDS are less effective in dissolving the protein.

The studies of Pusztai (31, 32) on the effects of phenol on proteins served as a guide to the development of the following method for the solubilization of the membrane protein in so- lutions of guanidine hydrochloride and urea near neutral pH. Membrane protein powder (about 10 mg per ml) was suspended in 80% phenol and mixed intermittently at room temperature for 2 hours. Most of the powder dissolved but a small amount of gelatinous material remained. The solution was made 20% in acetic acid by the addition of glacial acetic acid and the gel immediately disappeared, leaving the solution clear and colorless. The solution was dialyzed into 20% acetic acid for 24 hours at room temperature and then into 5.8 M guanidine hydrochloride in 0.05 M phosphate buffer at pH 7.7. The solution remained clear throughout the dialyses. If desired, the protein could then be dialyzed into 8 M urea and the protein remained in solution. The protein precipitated in aqueous buffers in the absence of urea or guanidine.

Succinylation resulted in complete solubility of the membrane protein in the absence of dissociating agents. More than 99% of the free amino groups were succinylated by the technique described.

Number of Proteins in Membrane-The results of the NH*- terminal amino acid determinations on the total membrane protein are given in Table IV. The major NH&erminal ammo acids were glycine, alanine, glutamic acid, and leucine.

Smaller amounts of aspartic acid and serine were present in all determinations and in one instance a small amount of valine was detected, although this may be a result of trace contami- nation by hemoglobin.

Polyacrylamide gel electrophoresis resulted in multiple bands, although results depended strongly on the method used to prepare the gel (Fig. 3). The 7.5 y. gels in 8 M urea showed about eight bands. In all runs a slowly moving band is present sur- rounded by several faint bands. In the absence of urea in 7.5%

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TABLE IV NHt-terminal analysis of membrane protein

Amino acid Percentage of total end groups recovered’s b

Glutamic acid. . . . 26.0 Glycine............................... 23.7 Alanine............................... 19.7 Leucine............................... 13.0 Aspartic acid. 9.3 Serine................................ 6.7 Valine................................ 1.3

o Average of three determinations (&15yo). b Of these NH&erminal amino acids, 0.6 mole was recovered

per 106 g of membrane protein. If recoveries are the same as those determined by Stark and Smyth (18) then 0.73 mole of NHz-terminal amino acids is obtained from 106 g of membrane protein.

FIG. 3. Polyacrylamide gel electrophoresis of lipid-extracted membrane protein. Identical protein samples were studied on gels with varying characteristics. The gels were prepared (left to right) with 73% acrylamide with 8 M urea, 73’% acrylamide with- out urea, 50/e acrylamide without urea, 4% acrylamide without urea, and 10% acrylamide in 0.1% sodium dodecyl sulfate without urea.

gels smearing of the bands occurs with poor resolution. In both 4% and 5% gels (no urea) the majority of the staining was in one or two major peaks traveling just before the marker dye (bromphenol blue). Two very faint slowly moving bands were the only other bands present.

In 10% gels containing 0.1% SDS from 15 to 25 bands were obtained. The presence of 0.1 M @-mercaptoethanol in the sample had no effect on these results.

Size of Proteins in Membrane-Sedimentation velocity studies of the membrane protein were performed in 0.9% formic acid, 62% formic acid, unbuffered 1% SDS, and 5.8 M guanidine hydrochloride in 0.05 M sodium phosphate buffer at pH 7.0. Succinylated protein was also studied in 0.05 M phosphate buffer at pH 7.0. The results are shown in Table V and Fig. 4.

The succinylated protein in phosphate buffer and the un- modified protein in formic acid sedimented in two main classes. To determine whether both peaks represented protein material, sucrose gradients of the membrane protein were run in 0.9% formic acid. Tubes were assayed by ninhydrin reaction after alkaline hydrolysis. Both peaks were composed of protein. The results are shown in Fig. 5.

In SDS and guanidine HCl only the slowly moving peak is present.

A pellet was seen in some studies that probably represents the small percentage of insoluble protein present and some non- protein material. This pellet was not observed in sucrose density gradient studies.

Sedimentation equilibrium studies were performed in 1% SDS and in 5.8 M guanidine hydrochloride. The effective weight average molecular weight (MW) of the membrane protein was determined simultaneously at three different concentrations in a three-channel Yphantis cell. M, is presented as a function of protein concentration in Table VI. Apparent li;r, increases

TABLE V

Sedimentation coefkients of lipid-extracted membrane protein”

Solvent 5,o.w x 10’2 set

0.9% formic acid. .................. 10.7 and 1.7 62.0% formic acid. .................... 7.1 and 2.3 38.0% formic acid. ..................... 0.8 Succinylated protein in phosphate buffer 13.6 and 1.9 1% sodium dodecyl sulfate ........... 1.7 5.8 M guanidine hydrochloride ........ 1.0

a The protein concentration was 0.5 g/100 ml.

FIG. 4. Ultracentrifugal patterns obtained from lipid-extracted membrane protein in 0.9% formic acid (left) and 1% sodium dodecyl sulfate (right) at 59,780 rpm and 20°. Photograph was taken after about 1 hour.

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with decreasing protein concentration. This is characteristic of a heterogeneous system.

Accurate determination of the molecular weight range of the protein components is not possible by this method but some estimate of the molecular weight of the smallest component can be obtained. For the concentration range near 5 mg per ml four determinations of the molecular weight in 5.8 M guanidine hydrochloride averaged to 53,000 f 7,000 average deviation. r’ was taken as 0.02 less than that computed from the amino acid composition as suggested by Marler, Nelson, and Tanford (33). The average of six determinations in this concentration range in 1% SDS yielded 49,900 f 7,000 average deviation. The measured P in this solvent was used in these calculations.

The results of the measurement of v are shown in Fig. 6. The calculated value of f from the equilibrium profiles is 0.78. The partial specific volume as calculated from the amino acid composition is 0.74 and is not changed by correcting for the known amounts of lipid and carbohydrate present.

Results of gel filtration chromatography of the total membrane protein are given in Fig. 7. The elution profiles of bovine serum albumin and lysozyme are also shown. The membrane protein divides into three well separated peaks. The lowest molecular weight component represents about 70% of the total protein added to the column and elutes near bovine serum albumin (molecular weight 68,000) and between it and lysosyme (molec

1.1

1.0

0.9

3, 0.8

5 0.7

5; 06

d 0.5

d 0.4

0.3

0 4 8 12 16 20 24 28 32 FRACTION NUMBER

FIN. 5. Sucrose density gradient (20 to 5y0 sucrose) centrifuga tion of lipid-extracted membrane protein in 0.9% formic acid at 50,000 rpm and 4O for 6% hours. Fractions were assayed by nin- hydrin reaction after alkaline hydrolysis. Sedimentation coeffi- cients are estimates obtained by comparing the position of the pro- tein peaks with those of molecules of known sedimentation coefficient centrifuged under similar conditions.

TABLE VI Effect oj varying initial protein concentration on apparent molecular

weight of membrane protein’ I

Initial protein concentration Measured apparent weight average molecular weight

w/ml

5.0 60,100 2.5 75,585 1.25 88,934

o Measured by the Yphantis sedimentation equilibrium method (21).

t-2 (cm21

FIG. 6. Sedimentation equilibrium analysis of lipid-extracted membrane protein in 1% solutions of sodium dodecyl sulfate pre- pared with either Hz0 or DZO. Centrifugation was performed at 37,020 and 20’. Fringe displacement is plotted on a logarithmic scale as a function of the square of the distance from the axis of rotation.

.5c’T”

0 20 40 60 80 100 120 140 160 180 230

FRACTION NUMBER FIG. 7. Gel filtration chromatography of lipid-extracted mem-

brane protein (dark line) on Sepharose 4B in unbuffered 1% sodium dodecyl sulfate. Bovine serum albumin and lysosyme were chromatographed on the same column under identical conditions. Fractions were analyzed by ninhydrin reaction after alkaline hy- drolysis.

ular weight 14,400). The protein from this peak was pooled, concentrated, and rerun on the same column. It yielded the same peak and did not aggregate to form the higher molecular weight components in the orignial mixture.

DISCUSSION

The red blood cell was chosen for the study of membrane chemistry for two reasons.

The membranes of the red blood cell are very similar fumc-

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tionally to the membranes of other cells. The mature erythro- cyte actively transports sodium and potassium ions (34), ex- hibits the facilitated transport of sugars (35, 36), and has a wide range of antigenic determinants (37). These functions are all present in the hemolyzed red blood cell (3537).

The other reason is the ease with which one can obtain large quantities of membrane free of other intracellular material. Although some enzymes are probably present in our membrane preparations (38, 39), they represent a very small fraction of the total protein present.

A major problem which confronted us in this study was the presence of lipid in the red blood cell membrane (see Table I). Previous investigators have isolated fractions of the membrane proteins either after partial lipid extraction of the membrane or after disruption of the intact membrane (40-48). Most of these preparations of the membrane proteins were heterogeneous in size or electrophoretic mobility. However, it is not clear whether the heterogeneity is related to the protein component or to the lipid part of these lipoprotein aggregates.

Indeed, in early studies we attempted to solubilize the mem- brane protein in the presence of lipid and we found that sonic disruption2 solubilized about 70% of the total membrane pro- tein. The solubilized lipoprotein appeared large in ultra- centrifugal studies, could not be dissociated by urea, and was excluded by all gel filtration resins. Electron microscopic photographs of the lipoprotein in solution showed the presence of spherical structures 150 to 450 A in diameter.

‘Because of these problems arising from the study of the membrane protein and lipid together in solution we chose to study the lipid-extracted membrane. Although lipid extraction very probably changes the tertiary structure of the membrane proteins, this approach may lead to a greater understanding of the chemical nature of the protein found in the membrane.

Solubility of Lipid-extracted Membrane Protein-Four different methods have been developed for solubilizing the membrane protein preparations. The solvent systems vary widely in their general nature and interpreted together lessen the pos- sibility of artifacts arising from the qualities of any single solvent.

Formic acid, 88%, solubilizes over 97% of the membrane protein. The protein remains in solution when dialyzed into 1% formic acid. The many disadvantages of working at this extreme pH limit the usefulness of this method.

Sodium dodecyl sulfate solubilizes over 98% of the membrane glycoprotein. SDS has been shown to be a powerful dissociation agent for proteins (49) and has been used by other workers to solubilize the intact membranes of both mammals and bacteria (7, 44, 50, 51). Recent studies have shown that hydrophobic interactions between SDS and the hydrophobic areas on proteins are largely responsible for its protein binding (52). The large number of hydrophobic residues present in the extracted protein available to solvent in the absence of lipid makes SDS a logical choice for the solubilization of the extracted membrane protein.

Once bound to protein SDS is difficult to remove completely. Because of its charge, the presence of SDS tightly bound to protein complicates attempts to separate proteins by ion ex- change chromatography. The pore sizes of gel filtration resins

* An ultrasonic disintegrator (Measuring and Scientific Equip- ment, Ltd., London, England) was used for two 3-min periods at 2o”.

are radically altered by 10/O SDS, also limiting the use of this technique for protein separation.3

Complete solubilization is achieved by succinylation of the membrane protein. In addition to the sedimentation studies reported here this technique has been used to prepare samples for ion exchange chromatography. Although the assays de- scribed indicate complete succinylation of all free amino groups it is possible that small numbers are not succinylated. A heterogeneous population of molecules might thus be produced, separating from one another on ion exchange resins, but whose heterogeneity is due to incomplete succinylation. We have separated succinylated protein into several fractions on ion exchange resins and are investigating the above possibilities.

The technique described here for the solubilization of the total membrane protein in urea and guanidine hydrochloride solutions is the first description of a technique for solubilizing the mem- brane protein at neutral pH in the absence of detergents. This method solubilizes the total extracted membrane powder. Although the protein cannot be directly solubilized in urea or guanidine hydrochloride the proteins can be made soluble in these solvents by first solubilizing in phenol. The highly dis- sociating effects of urea and guanidine on proteins and the ability to perform ion exchange chromatography and electro- phoresis in concentrated urea solutions make these solvents promising ones for the separation of membrane proteins (53).

Nature of Membrane Protein-The membrane protein prepara- tion contains appreciable carbohydrate after lipid extraction, confirming the view that the membrane has a significant amount of glycoprotein (37). Knowledge of the distribution of the carbohydrate on the membrane proteins awaits separation of the individual protein components of the membrane.

The amino acid composition of the membrane protein reveals a high percentage of amino acids with long nonpolar side chains. This is to be expected if the binding of lipid to membrane protein is in part by hydrophobic interactions. There is a small excess of acidic amino acids (13.76% as opposed to 12.18% basic amino acids). It has been shown, however, that the strong anionic character of the membrane is due to negatively charged sialic residues present in the membrane (54).

Although the number of proteins in the membrane is unknown, our results indicate the presence of multiple protein species in the membrane. The danger of using polyacrylamide gel electrophoresis to determine the number of protein components is emphasized by the dependence of the number of protein bands obtained on the individual characteristics of the gel used. The presence of multiple NHz-terminal amino acids shows, however, that many polypeptide chains are present. In assays for neutral and acidic NHz-terminal amino acids six different end groups are consistently found. Many more chains may be present since more than one chain may have the same end group and some end groups may be blocked. The values given in Table IV do not necessarily represent the relative amounts of the different chains since the yields of the amino acids by the assay used are different (18).

Additional evidence that several protein components are present in the membrane is shown in the preliminary observa- tions on the chromatographic separation of the protein com- ponents (Fig. 7).

Both succinylated protein and protein in formic acid fall into

* Unpublished results.

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1992 Protein of Human Erythrocyte Membranes. I Vol. 243, i\-o. 8

two size classes as revealed by sedimentation velocity studies. The two size classes have sp,, values near 1.5 S and 10 S. Sucrose gradient studies in formic acid (Fig. 5) show that both classes contain protein and that 86% of the protein is in the light fraction. In both SDS and guanidine hydrochloride solu- tions only the smaller size class is seen. This suggests strongly that the 10 S fraction represents aggregates of smaller molecular weight material. The aggregates are not present in the latter two solvents.

The interpretation of sedimentation equilibrium studies of heterogeneous systems is difficult. The effects of heterogeneity and nonideal behavior in solution tend to cancel each other and often yield daba that appear to be consistent with a monodisperse solution (see Fig. 5). The linearity seen in Fig. 6 is thus mis- leading. The contribution of small molecular weight species varies with the initial protein concentration used and hetero- geneity of the sample may be shown by varying the initial con- centration of the protein (21). As is seen in Table VI the ap- parent weight average molecular weight varies appreciably with concentration as expected for a heterogeneous system.

On the basis of the studies presented some of the proteins present have molecular weights near 50,000 in both SDS and guanidine hydrochloride. These solvents are known to unfold protein chains. Sedimentation velocity studies of unfolded proteins in this weight range are known to yield s~,~ values of 1.5 to 2.0 S (49). The s~,~ values found in the studies reported here on the membrane proteins fall in this range and are con- &tent with the above interpretation.

Additional evidence that most of the membrane protein is in the molecular weight range near 50,000 results from chromato- graphic studies in SDS (Fig. 7). The major protein peak rep- resents 70% of the total protein added to the column and falls between bovine serum albumin (mol wt 68,000) and lysozyme (mol wt 14,400). These membrane peaks represent different proteins as evidenced by the fact that samples from the individual peaks rechromatograph yielding only the peak from which they were taken. Polyacrylamide gel electrophoresis of each peak results in a set of bands different from that of any other peak.

The protein in the membrane of the human erythrocyte thus appears to be a heterogeneous collection of molecules. The molecular weights of the major protein components fall in the range normal for that of most globular proteins. Speculation as to the organization, functions, and modes of action of the mem- brane awaits the separation and study of the individual protein components. These studies are now in progress.

Acknowledgment--We would like to thank Professor John T. Edsall for his advice and encouragement throughout the present work.

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Steven A. Rosenberg and Guido GuidottiSOLUBILIZATION, AND PARTIAL CHARACTERIZATION

The Protein of Human Erythrocyte Membranes: I. PREPARATION,

1968, 243:1985-1992.J. Biol. Chem. 

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