Concentration of proteins using carboxymethyldextran

ANA1

YTICAL

98,

HIOCHFMISTRY

Concentration ANTHONY

353-357

(1979)

of Proteins R.

Using Carboxymethyldextran

TORRES

AND

ELBERT

Received

January

A.

PETERSON

19, 1979

Carboxymethyldextran was used to displace several proteins from DEAE-cellulose in highly concentrated form. and the concentrated protein fractions were examined directly by gel electrophoresis. The concentration of DNA-dependent RNA polymerase II demonstrates the application of the method to a labile enzyme. The concentration of goat y-globulin by sharp elution from CM-cellulose with carboxymethyldextran is also described.

chloroacetic acid and lithium hydroxide monohydrate were Fisher products. DEAEcellulose (DE-52) and CM-cellulose (CM-52) were supplied by Whatman, Inc. Ovalbumin (Grade VI) and ceruloplasmin (human type III) were furnished by the Sigma Chemical Company. Miles Laboratory supplied the P-lactoglobulin A. Prrpmrcrtion of CM-dextrun. The procedure was a modification of one previously reported ( 1), which should be consulted for important details. In the present case, the reaction mixture consisted of 18 g of monochloroacetic acid, 10 g of water, 10 g of dextran (median M, = lO,OOO), and 14 g of LiOH.H,O. A 150-ml bath of cold water was used to absorb some of the heat of neutralization when the alkali was added, but the temperature of the mixture was allowed to rise slightly to hasten solution of the excess LiOH.H,O. The viscous, bubblefilled mixture was allowed to stand, sealed, for 72 h at 23”C, then 14 ml of concentrated HCI was added, followed by 30 ml of methanol. A precipitate resulting from the addition of methanol redissolved on stirring, and the clear solution was poured slowly in a thin, continuous stream, into 700 ml of rapidly stirred absolute ethanol. Stirring was continued for 20 min more, then the precipitate was filtered, washed with ethanol,

Concentrated proteins in low salt solutions are required for some biochemical procedures. Methods commonly used for concentrating proteins include ultrafiltration, dialysis followed by lyophilization, and precipitation. Although these methods are useful in many applications, losses are often high when small amounts of protein are to be concentrated. Such losses can usually be avoided by adsorbing the protein on a small ion-exchange column and then sharply eluting it witlh a strong salt solution. This method can achieve substantial concentrations; however, the salt level in the concentrated solution is usually sufficiently high to interfere with evaluation by electrophoresis. This paper describes a method that utilizes a carboxymethyldextran (CM-dextran) with a high content of carboxyl groups to displace proteins (1) from DEAEcellulose. Since little salt is required, the highly concentrated proteins can be examined directly by gel electrophoresis. A procedure that employs a CM-dextran solution to concentrate proteins by elutiorz from CM-cellulose is also described. MATERIALS

AND METHODS

Dextran T 10 (lot No. 0094) was obtained from Pharmacia Fine Chemicals. Mono353

0003-2697/79/140353-05%0'.00/0 Copyright ‘d 1979 by Academic Prr%. Inc. All rIghI\ of rrprduct,on rn any lonn rererved.

354

TORRES

AND

and dried on the filter to a loose powder (about 18 g). This was redissolved in a mixture of 5 ml of concentrated HCI and 15 ml of methanol. Another 45 ml of methanol was added, then 60 ml of acetone was stirred in slowly. The clear solution was poured in a thin, continuous stream into 1200 ml of rapidly stirred acetone to produce a finely divided precipitate, which was filtered after 20 min of stirring and washed with acetone until the filtrate was no longer acid to Congo Red paper. The product was dried to a loose powder (about 16 g) in a stream of warm air. It was then dissolved in 30 ml of water and the pH was adjusted to approximately 11 with 4 M KOH (about 14 ml). The pH drifted rapidly downward so small additions of 4 M KOH were made until the drift at pH 11 was very slow. The solution was then added, dropwise, to 900 ml of rapidly stirred absolute ethanol to obtain a granular precipitate. The latter was filtered, washed with ethanol, and pulled dry (about 18 g). It was again dissolved in 30 ml of water and precipitated by dropwise addition to 900 ml ethanol. After the precipitate was filtered, washed with ethanol, and pulled dry in warm air, it was allowed to equilibrate with room air to constant weight. Of the 17 g of airdried product, 7 to 8% represented water that could be removed by desiccation in ~UC‘UO over anhydrous CaCl,. A 3.0% stock solution in water was prepared and clarified by filtration before use. Its chloride content was 0.002 M. Dividing the absorbance of the solution at 220 nm by the refractive increment (AN = 11,,,,- II,, .t,,) provides a convenient indicator that is proportional to carboxyl group content (1). This ratio was 560 for the potassium salt of the CM-dextran, corresponding to about 1000 for the acid form.

PETERSON

column to hold a disk of porous polyethylene in place. After the adsorbant had been packed under 10 psi of air pressure, a similar section of Tygon tubing containing a porous disk and filled with buffer to exclude air was fitted over the top of the column. A downward flow was used in all experiments. Prior to measuring absorbance at 280 nm. the 0.22-m] fractions were diluted with 1.0 ml of water. After spectrophotometry, lOO~1 portions were removed from each of the diluted fractions and pooled for protein determinations by the Lowry method (2). A vertical slab apparatus was used for the electrophoresis of the fractions in the discontinuous polyacrylamide gel system of Davis (3). Each sample applied to the gel was a mixture of 7 ~1 of 30% sucrose and 43 ~1 of diluted effluent fraction. After electrophoresis on the 75 x 175 x 2-mm at 200 V and 40 mA for about 2 h. the gel was stained with amido schwarz 10B. RESULTS AND DISCUSSION

A sample containing 10 mg of p-lactoglobulin A in 50 ml of 10 mM sodium phosphate, pH 7.5, was pumped into a 150-~1 column of DEAE-cellulose at room temperature. The adsorbed protein was then displaced from the column by a 1% solution of CM-dextran in the same buffer, resulting in a 96% recovery of the protein in about 1 ml (Fig. 1A). Identical conditions were used to concentrate ovalbumin and ceruloplasmin (Figs. 18 and C) from dilute solutions (10 mg in 50 ml) to 1 and 1.5 ml, respectively, with recoveries of 95 and 93%. respectively. The presence of CM-dextran in some of the concentrated protein fractions did not interfere with the direct examination of the proteins by gel electrophoresis. The Chromutograph~ md electrophorrsis. Columns of 150 ~1 (27 x 2.7 mm) and 100 CM-dextran migrated in the gel more ~1 (18 x 2.7 mm) were made from sections rapidly than any of the proteins and could of l-ml disposable glass pipets (Corning be seen as a sharp refractile band at the Glass Works). A short section of Tygon tub- buffer discontinuity. The patterns obtained ing was stretched over the bottom of each by electrophoresis of the concentrated p-

CONCENTRATION

OF PROTEINS

USING

lactoglobulin A, ovalbumin, and ceruloplasmin (Figs. 2A, B, and C) showed that these proteins migrated at the same rate as unchromatographed standards. The minor components shown in the electropherogram were impurities in the original material and appeared when standards were used at sufficient concentration. The CM-dextran can also be used to concentrate proteins by elution from CMcellulose. Since the CM-dextran has no affinity for CM-cellulose, it cannot function as a displacer in the usual sense. yGlobulin was precipitated from goat serum, along with other proteins, by the addition of 0.75 ml of 36% sodium sulfate per milliliter of serum, and the precipitate was dialyzed exhaustively against 10 mM sodium

FIG. 1. Concentration of proteins. Displacement: A sample containing 10 mg of p-lactoglobulin A (A), ovalbumin (B), or ceruloplasmin (C) in 50 ml of 10 mM sodium phosphate, pH 7.5. was pumped at 5 ml/h into a 150.~1 column of DEAE-cellulose equilibrated with the same buffer. The adsorbed protein was then displaced with a 1% CM-dextran solution in that buffer. Elution: 10 mg of crude goat y-globulin (D) in 34 ml of 10 mM sodium Iphosphate, pH 6.0, containing 0.074 M glycine was pumlped at 5 ml/h into a 150-p] column of CM-cellulose equilibrated with the same buffer. About 8 mg of the protein was adsorbed, then it was sharply eluted with a 1% CM-dextran solution in the above buffer. All of these experiments were conducted at room temperature. The CM-dextran solution was pumped at 2.5 ml/h in every case, and 0.22-ml fractions were collected.

CARBOXYMETHYLDEXTRAN

355

phosphate, pH 7.6. The dialyzed protein (12 mg in 9.25 ml) was mixed with 2.5 ml of 1.0 M glycine and 22.5 ml of 10 mM sodium phosphate, pH 6.0, and centrifuged at 1OOOg for 20 min. The supernatant liquid (34 ml containing 10 mg of protein) was then applied at room temperature to a 150-~1 column of CM-cellulose equilibrated with 10 mM sodium phosphate, pH 6.0. About 8 mg of the protein was adsorbed. A 1% solution of CM-dextran in the same buffer eluted this sharply, resulting in the concentration of the protein to 1 ml, with a recovery of more than 90% of the adsorbed protein (Fig. 1D). Electrophoresis of the concentrated y-globulin in the high-pH system proceeded normally (although complexes with CM-dextran probably existed in the samples) and the migration rate was the same as that of a standard containing no CM-dextran (Fig. 2D). The concentrated yglobulin, however, appeared to have none of the other proteins that contaminated the original precipitate, though they should have been readily visible at the concentration used. Presumably they were lost with the 20% of the original protein that was not adsorbed by the CM-cellulose under the conditions employed. The concentration of DNA-dependent RNA polymerase 11 (Fig. 3) provides an example of the application of the displacement method to a labile enzyme. Two colleagues (W. H. Kastern and K. P. Mullinix) involved in the purification of this enzyme from chicken liver have found the method useful because of the need to concentrate the enzyme rapidly before proceeding from one step in the purification to the next, while avoiding high salt concentrations, which inactivate the enzyme. For the example illustrated, a lo-ml sample containing polymerase II activity in 0.5 mg of protein recovered from a Sepharose 6B column was pumped at 5 ml/h into a lOO-~1 column of DEAE-cellulose at 4°C. Fractions containing 60 ~1 were collected during application of the 1% CM-dextran solution

356

TORRES

A

AND

PETERSON

B

c hJ/.

FIG. 2. Electrophoresis of chromatographic fractions. Samples (43 ~1) of effluent fractions from the experiments illustrated in Fig. 1 were subjected to electrophoresis in a vertical 7.5%’ polyacrylamide gel, with migration toward the anode at the bottom. The letters correspond to the experiments shown in Fig. 1. S designates standards: 40 yg of P-lactoglobulin A. 40 pg of ovalbumin. 25 pg of ceruloplasmin, and 3.5 pg of crude y-globulin.

at 2.5 ml/h. All solutions, including those in the columns as well as the CM-dextran solution, contained 2.5 mM Tris chloride (pH 8.0), 2 mM dtthtothreitol, 20 Fg/ml ot phenylmethanesulfonyl fluoride, 10% glycerol. and 50 mM ammonium sulfate. Assay of the sample from the Sepharose column and the fractions from the concentrating column for DNA-dependent RNA polymerase II activity (4) showed that the IOml enzyme sample had been concentrated to about 0.3 ml, wtth recovery of about 85% of the activity. Although the displaced protein moves ahead of the CM-dextran on the column, CM-dextran will inevitably be in some of the protein-containing fractions. This may inhibit certain enzymes, especially those that require a metal ion. Therefore, before concentration of an enzyme is attempted, its tolerance for CM-dextran should be determined, with a vtew to lowering the concentration of the CM-dextran employed, if necessary, or to reversing any inhibition with added metal ion. In the case of the polymerase, the use of a I’% CM-dextran solution did not interfere with the assay. The CM-dextran employed in these ex-

10

If., \ 00

1 0.2

/ 0.4 VOLUME

0.6

I 0.8

(ml)

FIG. 3. Concentration of DNA-dependent RNA polymerase II by displacement. A IO-ml fraction containing polymerase II activity from a Sepharose 6B column was applied to a lOO-~1 DEAE-cellulose column, at 4°C. Following the sample application, a 1% CM-dextran solution was pumped into the column at 2.5 ml/h. and 60-p] fractions were collected. All solutions contained 10% glycerol. 50 mM ammonium sulfate, 2 mM dithiothreitol, 20 &ml of phenylmethanesulfonyl fluoride. and 25 mM Tris chloride, pH 8.0. The ordinate shows the amounts of ]:‘H]UTP polymerized by enzyme in fractions.

CONCENTRATION

OF

PROTEINS

periments was a highly substituted preparation with an average of almost one carboxymethyl group for every glucose residue. Its high affinity for DEAE-cellulose provides a Ibasis for believing that it can displace almost any protein from that adsorbent. However, CM-dextran preparations having somewhat lower carboxymethyl content. and more easily purified because their free acild forms are insoluble in ethanol, would probably be suitable for displacing most proteins. The preparation of such CMdextran having A,,,,/Atl values of 750-800 in the acid form, 400 in the salt form, is described in an earlier paper ( 1).

USING

357

CARBOXYMETHYLDEXTRAN

ACKNOWLEDGMENTS The authors wish to thank Drs. W. H. Kastern and K. P. Mullinix for permitting the data on the concentration of DNA-dependent RNA polymerase II to be presented in this paper. The technical assistance of Mr. Hugh Foster is gratefully acknowledged.

REFERENCES I. Peterson.

E. A. t 1978) And.

Biochrm.

90. 767-

784.

2. Lowry, 0. H., Rosebrough. N. J.. Far-r. Randall. R. J. (1951) J. Biol. Chrrrr. 27s. 3. Davis.

.--

B. J. (1964)Ann.

hr. Y. Awd.

G.. and Cambon, L-25.

P. (1976)&r.

A. L.. and 193, 265-

SC;. 121,404-

4Ll.

4. Krebs, 61,

J. Bioc,helrr.