ANALYTICAL
67. 55-65
BIOCHEMISTRY
Removal
of Sodium
(1975)
Dodecyl
Sulfate
from
Proteins1
GEORGE P. TUSZYNSKI AND LEONARD WARREN Department
University
of Therapeutic of Pennsylvunicr.
Received
May
29, 1974:
Research. Philadelphia. accepted
School of Medicine. Pennsylvania 19174 February
3, 1975
A convenient and relatively simple electrodialysis method for the removal of sodium dodecyl sulfate (SDS) from proteins is described. Six samples can be processed simultaneously. The kinetics of removal of SDS from proteins by equilibrium dialysis and electrodialysis have been studied,
INTRODUCTION
In the course of purifying membrane proteins from normal and malignant baby hamster kidney cells grown in tissue culture, it became necessary to utilize the excellent solubilizing properties of sodium dodecyl sulfate (SDS). In addition to powerful solubilizing properties, the use of this detergent afforded us a means of separating proteins according to molecular weight by the well-known method of SDS-polyacrylamide electrophoresis (1). Inevitably, however. in order to carry our purification process further, we hoped to utilize methods such as isoelectric focusing and electrophoresis in the presence of urea that separate proteins according to chavge. These procedures necessitate the removal of SDS. We report here the results of dialysis and electrodialysis of SDS from proteins including bovine serum albumin (BSA) and membrane proteins. MATERIALS
AND
METHODS
Muterids. Urea (reagent grade), tris(hydroxymethyl)aminomethane (Tris) base (reagent grade), sodium mono- and dibasic phosphates (reagent grade) were purchased from Fisher Scientific Co. (Pittsburgh, PA). Mercaptoethanol (reagent grade) was purchased from Sigma Chemical Co. (St. Louis, MO). SDS (99% purified) was purchased from Gallard-Schlesinger Chemical Mfg. Corp. (Carle Place, L.I., NY). Labeled SDS (35S-SDS) (1 mCi/15.8 mg, 99.9%) was purchased from New England Nuclear (Boston, MA). BSA, ovalbumin and cytochrome ( 1 The work described in this paper was supported by grants from the American Cancer Society, No. PRP-28 and BC16C, the U.S. Public Health Service, No. I ROI CA 13992-02 and 1 PO1 CA 14499-01, and by a postdoctoral fellowship (G.P.T.) from the National Institutes of Health No. 1 F02AMS5449-0 I. 55 Copyright
0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
56
TUSZYNSKI
AND
WARREN
Plexi
-'/l/
gasket llulose membrane
FIG.
1. Top,
entire
electrodialysis
apparatus.
Bottom,
diagram
of cell
compartments.
REMOVAL
OF
SDS
FROM
PROTEINS
57
were purchased from Nutritional Biochemicals Corp. (Cleveland, OH). Deionized water was used throughout. Dialysis tubing (12,000-M, exclusion limit) and collodion bags @-ml capacity, 20,000-M, exclusion limit) were purchased from A. H. Thomas (Philadelphia, PA). Equipment. An Intertechnique scintillation counter (Westwood, NJ) was used for counting samples. A Buchier power source (Lee, NJ) was used for the electrodialysis experiments. Electrodialysis was carried out in a modified Canalco, Inc. (Rockville, MD) gel destainer (#1801). The center partition (between the positive and negative electrodes) where the gels are normally placed was removed and replaced by a cell compartment (Fig. 1). The dialysis cells were cut in 3/l 6-in. Plexiglas. Dialysis tubing (1 3/S-in. diam) cut open and stretched across the cells served as membranes. Gaskets were cut from l/32-in. silicone rubber. Natural-color silicone rubber that does not contain dye is preferable. Kinetic equilibrium dialysis experiments were performed by suspending collodion bags in 300-ml Erlenmeyer flasks. The bags were suspended from 14/20 ground-glass joints fitted in #6 rubber stoppers. Each glass joint was covered with a short piece of tight-fitting Tygon tubing. The collodion bags were slipped onto the Tygon-tubing sleeve producing a firm leak-proof joint. This apparatus was convenient because it could be sealed to prevent evaporation by stoppering the top of the glass joint and can provide a port for sampling. The solutions were continuously mixed with magnetic stirrers in the equilibrium dialysis experiments. Additionally, kinetic equilibrium experiments were performed by suspending dialysis bags into 300-ml Erlenmeyer flasks. Methods. Normal BHK,,/C,, and Rous sarcoma virus-transformed C&/B, baby hamster kidney cells were grown in tissue culture as described elsewhere (2). Crude 12,000-g pellet membrane fractions used in this study were prepared from these cells (3). SDS electrophoresis of these fractions indicated at least 40 different proteins ranging in molecular weight from 250,000-10,000. A single C,,/B, membrane protein of approximately 30,000 daltons was isolated from SDS gels (4). This protein was labeled in its amino acids by 14C-labeled amino acid mixtures. A stock 35S-SDS solution having a specific activity of 1 mCi/ml was prepared by dissolving 15.8 mg of labeled SDS in 1 ml of distilled water. Aliquots of this solution were added to nonradioactive SDS and SDSprotein solutions so that 5 to 10 ~1 gave I O,OOO-70,000 cpm. Buffers for dialysis experiments were as follows: A, 5 M urea, 1 mM Tris hydrochloride, pH 7.7, 1% mercaptoethanol; B, 1 mM Tris hydrochloride, pH 7.7, 1% mercaptoethanol; C, 0.1 M sodium phosphate, pH 6.8, 1% mercaptoethanol. All protein-SDS solutions containing 35S-SDS were prein-
58
TUSZYNSKI
AND
WARREN
cubated for 2 days at room temperature to insure complete complexation with protein (5). A typical kinetic, equilibrium dialysis experiment (Fig. 2) was performed by adding 3 ml of buffer containing 0.05% BSA, 0.1% SDS with label to a collodion bag or dialysis tubing suspended in 300 ml of buffer. While the outer solution was stirred with a magnetic stirrer, samples of 10 ~1 were withdrawn periodically and counted. In experiments in which the outer solution was repeatedly changed (Table 1) one ml of protein“5S-SDS was dialyzed against 125 ml of buffer. The exterior solution was stirred with a magnetic stirrer. Electrodialysis experiments were performed by injecting the desired SDS-protein solution containing 35S label through the sample port of each cell. Buffer was added to the outer compartments to a level equal to that inside the sample cells. Solutions containing 0.1% SDS and 0.05% BSA in buffers A and C and solutions containing 0.2% SDS, 0.05% isolated C,,/B, membrane protein, C,,/B, membrane fraction and BHK2JC13 membrane fractions in buffer A were electrodialyzed. Control experiments containing SDS but no protein were performed in the same manner. In the BSA experiments, 5 ml of solution were placed in each cell. For the membrane protein experiments, 1 ml was used. Electrodialysis was performed at a constant current of 20 mA and 20 V. Voltage remained constant throughout the experiment. At periodic intervals, the power was turned off and duplicate samples (5-10~1) were withdrawn. Duplicate samples varied less than 5%. SDS efflux kinetics were analyzed according to standard procedures (6). Logarithmic plots were constructed by subtracting the value of the remaining “5S counts per minute at time infinity from the value determined at each interval. In the case of electrodialysis 35S counts at time infinity is zero, while for the kinetic equilibrium experiments a finite endpoint (4.7 moles of SDS-BSA) was subtracted from the radioactive value at each count-per-minute time point. Equilibrium conditions were achieved in the equilibrium kinetic experiments. The endpoints obtained with no changes of the outer solution after several days of dialysis equaled the value of 4.7 moles of SDS-BSA predicted from the dilution factor of 1 to 100 utilized in these experiments. The pH of the solutions remained constant in the course of the experiments with the exception of the electrodialysis experiments in buffers A and B where the pH increased 0.3 units over 6 hr. RESULTS Equilibrium
Dialysis
of SDS
Membrane composition had no apparent effect on the SDS dialysis rates. Collodion bags composed of cellulose nitrate and dialysis tubing
REMOVAL
OF
SDS
FROM
59
PROTEINS
104
,472
ld
4.72
0
2
4
6 HOURS
8
10
12
FIG. 2. Plot of A cpm of ?S-SDS remaining in the dialysis chamber against time for electrodialysis of SDS in buffer A with (0-O) and without (O-O) BSA: and for equilibrium dialysis in buffer A with (A-A) and without (n--a) protein. The scale on the right shows the number of moles of SDS per mole of BSA remaining in the chamber. Half times of the curves are given in Tables 1 and 2. Solutions contained 0.05% BSA and 0.1% SDS. A cpm of :Y3-SDS is defined as cpm of ?S-SDS at any time minus cpm of YS-SDS at time infinity (see Methods). The endpoint for the equilibrium dialysis experiments was 4.7 moles of SDS/mole of BSA, while that for electrodialysis was essentially zero moles of SDS/mole of BSA. All counts were corrected for background.
composed of cellulose acetate were both used in the kinetic equilibrium experiments and gave the same SDS efflux rates. The kinetics of SDS efflux from SDS-BSA solutions and controls at room temperature appear to be pseudo first order processes. Log plots of the counts of 35S-SDS remaining in the dialysis bag as a function of time are straight lines (Fig. 2). After 4 days of dialysis with changes every 24 hours less than 1 mole of SDS could be detected per mole of BSA (Table I). Protein slightly decreases the rate of SDS dialysis except when phosphate buffer was used where control is 1.5-fold faster. Solvent apparently influences the rate of SDS dialysis. Increased solvent polarity tends to increase the rate of SDS dialysis. SDS dialysis rates in buffer C are approximately 1.3-fold faster than in buffers A and B (Table 1). The enect of temperature on dialysis rates was not extensively stud-
60
TUSZYNSKI
EQUILIBRIUM
DIALYSIS
AND WARREN
TABLE 1 OF SDS AT
ROOM
TEMPERATURE
[SDS]/[BSA]* Sample” Buffer Buffer Buffer Buffer Buffer Buffer
A A-BSA B B-BSA C C-BSA
Ionic Strength 1.0 1.0 1.0 1.0
(M)
x 10-s x 10-s x 10-Z x 10-a 0.20 0.20
25 Hr
48 Hr
72 Hr
96 Hr
fliz WC
18 15 18 23 3.0 11
2.0 4.0 6.0 7.0 2.0 Cl.0
1.0 2.0 3.0 3.0 1.0 Cl.0
‘cl.0
67. 55-65
BIOCHEMISTRY
Removal
of Sodium
(1975)
Dodecyl
Sulfate
from
Proteins1
GEORGE P. TUSZYNSKI AND LEONARD WARREN Department
University
of Therapeutic of Pennsylvunicr.
Received
May
29, 1974:
Research. Philadelphia. accepted
School of Medicine. Pennsylvania 19174 February
3, 1975
A convenient and relatively simple electrodialysis method for the removal of sodium dodecyl sulfate (SDS) from proteins is described. Six samples can be processed simultaneously. The kinetics of removal of SDS from proteins by equilibrium dialysis and electrodialysis have been studied,
INTRODUCTION
In the course of purifying membrane proteins from normal and malignant baby hamster kidney cells grown in tissue culture, it became necessary to utilize the excellent solubilizing properties of sodium dodecyl sulfate (SDS). In addition to powerful solubilizing properties, the use of this detergent afforded us a means of separating proteins according to molecular weight by the well-known method of SDS-polyacrylamide electrophoresis (1). Inevitably, however. in order to carry our purification process further, we hoped to utilize methods such as isoelectric focusing and electrophoresis in the presence of urea that separate proteins according to chavge. These procedures necessitate the removal of SDS. We report here the results of dialysis and electrodialysis of SDS from proteins including bovine serum albumin (BSA) and membrane proteins. MATERIALS
AND
METHODS
Muterids. Urea (reagent grade), tris(hydroxymethyl)aminomethane (Tris) base (reagent grade), sodium mono- and dibasic phosphates (reagent grade) were purchased from Fisher Scientific Co. (Pittsburgh, PA). Mercaptoethanol (reagent grade) was purchased from Sigma Chemical Co. (St. Louis, MO). SDS (99% purified) was purchased from Gallard-Schlesinger Chemical Mfg. Corp. (Carle Place, L.I., NY). Labeled SDS (35S-SDS) (1 mCi/15.8 mg, 99.9%) was purchased from New England Nuclear (Boston, MA). BSA, ovalbumin and cytochrome ( 1 The work described in this paper was supported by grants from the American Cancer Society, No. PRP-28 and BC16C, the U.S. Public Health Service, No. I ROI CA 13992-02 and 1 PO1 CA 14499-01, and by a postdoctoral fellowship (G.P.T.) from the National Institutes of Health No. 1 F02AMS5449-0 I. 55 Copyright
0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
56
TUSZYNSKI
AND
WARREN
Plexi
-'/l/
gasket llulose membrane
FIG.
1. Top,
entire
electrodialysis
apparatus.
Bottom,
diagram
of cell
compartments.
REMOVAL
OF
SDS
FROM
PROTEINS
57
were purchased from Nutritional Biochemicals Corp. (Cleveland, OH). Deionized water was used throughout. Dialysis tubing (12,000-M, exclusion limit) and collodion bags @-ml capacity, 20,000-M, exclusion limit) were purchased from A. H. Thomas (Philadelphia, PA). Equipment. An Intertechnique scintillation counter (Westwood, NJ) was used for counting samples. A Buchier power source (Lee, NJ) was used for the electrodialysis experiments. Electrodialysis was carried out in a modified Canalco, Inc. (Rockville, MD) gel destainer (#1801). The center partition (between the positive and negative electrodes) where the gels are normally placed was removed and replaced by a cell compartment (Fig. 1). The dialysis cells were cut in 3/l 6-in. Plexiglas. Dialysis tubing (1 3/S-in. diam) cut open and stretched across the cells served as membranes. Gaskets were cut from l/32-in. silicone rubber. Natural-color silicone rubber that does not contain dye is preferable. Kinetic equilibrium dialysis experiments were performed by suspending collodion bags in 300-ml Erlenmeyer flasks. The bags were suspended from 14/20 ground-glass joints fitted in #6 rubber stoppers. Each glass joint was covered with a short piece of tight-fitting Tygon tubing. The collodion bags were slipped onto the Tygon-tubing sleeve producing a firm leak-proof joint. This apparatus was convenient because it could be sealed to prevent evaporation by stoppering the top of the glass joint and can provide a port for sampling. The solutions were continuously mixed with magnetic stirrers in the equilibrium dialysis experiments. Additionally, kinetic equilibrium experiments were performed by suspending dialysis bags into 300-ml Erlenmeyer flasks. Methods. Normal BHK,,/C,, and Rous sarcoma virus-transformed C&/B, baby hamster kidney cells were grown in tissue culture as described elsewhere (2). Crude 12,000-g pellet membrane fractions used in this study were prepared from these cells (3). SDS electrophoresis of these fractions indicated at least 40 different proteins ranging in molecular weight from 250,000-10,000. A single C,,/B, membrane protein of approximately 30,000 daltons was isolated from SDS gels (4). This protein was labeled in its amino acids by 14C-labeled amino acid mixtures. A stock 35S-SDS solution having a specific activity of 1 mCi/ml was prepared by dissolving 15.8 mg of labeled SDS in 1 ml of distilled water. Aliquots of this solution were added to nonradioactive SDS and SDSprotein solutions so that 5 to 10 ~1 gave I O,OOO-70,000 cpm. Buffers for dialysis experiments were as follows: A, 5 M urea, 1 mM Tris hydrochloride, pH 7.7, 1% mercaptoethanol; B, 1 mM Tris hydrochloride, pH 7.7, 1% mercaptoethanol; C, 0.1 M sodium phosphate, pH 6.8, 1% mercaptoethanol. All protein-SDS solutions containing 35S-SDS were prein-
58
TUSZYNSKI
AND
WARREN
cubated for 2 days at room temperature to insure complete complexation with protein (5). A typical kinetic, equilibrium dialysis experiment (Fig. 2) was performed by adding 3 ml of buffer containing 0.05% BSA, 0.1% SDS with label to a collodion bag or dialysis tubing suspended in 300 ml of buffer. While the outer solution was stirred with a magnetic stirrer, samples of 10 ~1 were withdrawn periodically and counted. In experiments in which the outer solution was repeatedly changed (Table 1) one ml of protein“5S-SDS was dialyzed against 125 ml of buffer. The exterior solution was stirred with a magnetic stirrer. Electrodialysis experiments were performed by injecting the desired SDS-protein solution containing 35S label through the sample port of each cell. Buffer was added to the outer compartments to a level equal to that inside the sample cells. Solutions containing 0.1% SDS and 0.05% BSA in buffers A and C and solutions containing 0.2% SDS, 0.05% isolated C,,/B, membrane protein, C,,/B, membrane fraction and BHK2JC13 membrane fractions in buffer A were electrodialyzed. Control experiments containing SDS but no protein were performed in the same manner. In the BSA experiments, 5 ml of solution were placed in each cell. For the membrane protein experiments, 1 ml was used. Electrodialysis was performed at a constant current of 20 mA and 20 V. Voltage remained constant throughout the experiment. At periodic intervals, the power was turned off and duplicate samples (5-10~1) were withdrawn. Duplicate samples varied less than 5%. SDS efflux kinetics were analyzed according to standard procedures (6). Logarithmic plots were constructed by subtracting the value of the remaining “5S counts per minute at time infinity from the value determined at each interval. In the case of electrodialysis 35S counts at time infinity is zero, while for the kinetic equilibrium experiments a finite endpoint (4.7 moles of SDS-BSA) was subtracted from the radioactive value at each count-per-minute time point. Equilibrium conditions were achieved in the equilibrium kinetic experiments. The endpoints obtained with no changes of the outer solution after several days of dialysis equaled the value of 4.7 moles of SDS-BSA predicted from the dilution factor of 1 to 100 utilized in these experiments. The pH of the solutions remained constant in the course of the experiments with the exception of the electrodialysis experiments in buffers A and B where the pH increased 0.3 units over 6 hr. RESULTS Equilibrium
Dialysis
of SDS
Membrane composition had no apparent effect on the SDS dialysis rates. Collodion bags composed of cellulose nitrate and dialysis tubing
REMOVAL
OF
SDS
FROM
59
PROTEINS
104
,472
ld
4.72
0
2
4
6 HOURS
8
10
12
FIG. 2. Plot of A cpm of ?S-SDS remaining in the dialysis chamber against time for electrodialysis of SDS in buffer A with (0-O) and without (O-O) BSA: and for equilibrium dialysis in buffer A with (A-A) and without (n--a) protein. The scale on the right shows the number of moles of SDS per mole of BSA remaining in the chamber. Half times of the curves are given in Tables 1 and 2. Solutions contained 0.05% BSA and 0.1% SDS. A cpm of :Y3-SDS is defined as cpm of ?S-SDS at any time minus cpm of YS-SDS at time infinity (see Methods). The endpoint for the equilibrium dialysis experiments was 4.7 moles of SDS/mole of BSA, while that for electrodialysis was essentially zero moles of SDS/mole of BSA. All counts were corrected for background.
composed of cellulose acetate were both used in the kinetic equilibrium experiments and gave the same SDS efflux rates. The kinetics of SDS efflux from SDS-BSA solutions and controls at room temperature appear to be pseudo first order processes. Log plots of the counts of 35S-SDS remaining in the dialysis bag as a function of time are straight lines (Fig. 2). After 4 days of dialysis with changes every 24 hours less than 1 mole of SDS could be detected per mole of BSA (Table I). Protein slightly decreases the rate of SDS dialysis except when phosphate buffer was used where control is 1.5-fold faster. Solvent apparently influences the rate of SDS dialysis. Increased solvent polarity tends to increase the rate of SDS dialysis. SDS dialysis rates in buffer C are approximately 1.3-fold faster than in buffers A and B (Table 1). The enect of temperature on dialysis rates was not extensively stud-
60
TUSZYNSKI
EQUILIBRIUM
DIALYSIS
AND WARREN
TABLE 1 OF SDS AT
ROOM
TEMPERATURE
[SDS]/[BSA]* Sample” Buffer Buffer Buffer Buffer Buffer Buffer
A A-BSA B B-BSA C C-BSA
Ionic Strength 1.0 1.0 1.0 1.0
(M)
x 10-s x 10-s x 10-Z x 10-a 0.20 0.20
25 Hr
48 Hr
72 Hr
96 Hr
fliz WC
18 15 18 23 3.0 11
2.0 4.0 6.0 7.0 2.0 Cl.0
1.0 2.0 3.0 3.0 1.0 Cl.0
‘cl.0
