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The removal of the neutral detergent Tween 20 from solubilized membrane proteins of Acholeplasma laidlawii and other proteins by agarose-suspension electrophoresis
ANALYTICAL
BIOCHEMISTRY
The Removal Solubilized
78, 171- 1st (1977)
of the Neutral Detergent Tween 20 from Membrane Proteins of Acholeplasma laidlawii and Other Proteins by Agarose-suspension Electrophoresis
GEORGE DRESDNER’AND HILDA CID-DRESDNER~ Membrane Group. Institute of Biochemistry, Biomedical Center, University Box 576, S-751 23 Uppsala, Sweden, and ZStructure Group, Wallenberg Uppsala University, Box 562, S-751 22 Uppsala, Sweden
of Uppsala, Laboratory,
Received June 29. 1976; accepted October 21. 1976 The neutral detergent Tween 20 can be detected in fractions from agarosesuspension electrophoresis by spectrophotometry and surface-tension measurements. A concentration of Tween 20 as low as 0.001% can be detected by surface-tension measurements. In runs performed at pH 8.0 in 0.1 M Tris-HCI, the detergent remains near the starting point. Using these conditions, the detergent can be removed from membrane proteins of Acholeplasma laidlawii solubilized by Tween 20 or from proteins such as human seturn albumin and horse heart cytochrome c to which detergent has been added. The method can be used on a IS-mg preparative scale.
When a neutral detergent is used for the separation and purification of membrane proteins, its removal from the purified fractions is often difficult (l), particularly when the sample has previously been concentrated. For instance, it cannot be removed by dialysis due to its large micellar size and its low critical micelle concentration which determines that a small difference in the concentration of free detergent will exist on both sides of the dialysis membrane (2-4). Successful removal of neutral detergents from proteins has been achieved by gel filtration (5) or by preparative polyacrylamide gel electrophoresis (6). In the latter case, the method has been used to remove the neutral detergent Tween 20 from purified membrane proteins of Acholeplasma laidlawii. But, with this technique, it is difficult to handle amounts of membrane proteins on a preparative scale (6,4). In the present work, agarose-suspension electrophoresis is shown to be a convenient technique for the removal of the neutral detergent Tween 20 from the solubilized membrane proteins of A. faidlnwii and from other proteins as well. r Present address: Biophysics Department, Arrhenius Laboratory. Stockholm University. S-104 05 Stockholm, Sweden.
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN Mw)3-2697
172
DRESDNER
AND CID-DRESDNER
MATERIALS
AND METHODS
Chemicals. All chemicals were reagent grade. Agarose powder was purchased from Bio-Rad Laboratories; Tween 20, quality SD, was purchased from Atlas Chemie, GmbH, Essen, Germany. Human serum albumin was from AB Kabi, Stockholm, Sweden. Horse heart cytochrome c from Sigma Chemical Co., St. Louis, Missouri was a gift of Dr. Per Lundahl . Agarose-suspension electrophoresis. Details of the technique have already been published (7,4). The agarose suspension was prepared in 0.1 M Tris-HCl, pH 8.0, and it contained 0.02% sodium azide. Spectrophotometric measurements. Absorbance measurements were performed in a PMQ III Zeiss spectrophotometer using quartz cuvettes with a l-cm pathlength. The absorbance of agarose-suspension electrophoresis fractions was measured in a PMQ II Zeiss spectrophotometer as already described (7). Absorption spectra were measured with a Model 25 Beckman recording spectrophotometer. Surface-tension measurements. A capillary-rise apparatus with a graduated capillary tube was used for surface-tension measurements. The apparatus was calibrated with distilled, filtered, and deionized water. Measurements were performed at 23°C. Analytical polyacrylamide gel electrophoresis. Electrophoresis in the presence of 0.02 M sodium dodecyl sulfate and 0.1 M Tris-HCI (pH 8.0) was performed on 0.6 x 10 x lO-cm gel slabs at room temperature. Details of the apparatus and technique have already been published (8). Gels with the following composition were used: T = 6%, C = 5% (8). With a voltage gradient of 10 V/cm, separation was achieved within 90 min. The gels were stained with Coomassie brilliant blue R 250. Sample concentration. In some cases, the samples were concentrated with a B- 1.5 Minicon miniconcentrator prior to analytical polyacrylamide gel electrophoresis. RESULTS
Two different techniques were used to detect the presence of detergent in fractions obtained from agarose-suspension electrophoresis. These were spectrophotometric and surface-tension measurements. The techniques were first standardized by performing measurements on Tween 20 solutions. Spectrophotometric
Measurements of Tween 20 Solutions
The absorption spectrum of Tween 20 is shown in Fig. 1A. gent solution was prepared in 0.1 M Tris-HCl, pH 8.0. Figure that part of the spectrum that is used in practice with the concentration commonly present in mixtures of solubilized
The deter1B shows Tween 20 membrane
REMOVAL
A
DETERGENT
.
173
C
.
12 .I
,” 2.0 c 2 0’ 2
8 LL
OF NEUTRAL
3
/
.
.I//
1.0
a
.I/
Kl
300
LOO
Wavelength
250
350
(nm)
10
30
Tween 20 concentrationp/.)
FIG. 1. Spectrophotometric measurements of Tween 20 solutions in 0.1 M Tris-HCI, pH 8.0. (A) Absorption spectrum of a 1% solution. (B) Absorption spectrum of a 3% solution; the figure shows that part of the spectrum that is used in practice. (C) Relationship between absorbance and concentration of a Tween 20 solution at different wavelengths: (I) 280. (2) 310, and (3) 350 nm.
proteins of A. laidlawii. Even when the spectrum shows a monotonous increase of absorbance towards shorter wavelengths, the detergent can be clearly distinguished from the background in fractions from agarosesuspension electrophoresis. Figure 1C shows that Tween 20 solutions follow Bourger- Lambert- Beer’s law within the range of concentrations examined. The maximal concentration studied was 40%. Its absorbance at 280 nm is larger than the maximally measurable absorbance of the instrument. But, measurements at lower wavelengths show that the relationship between Tween 20 concentration and absorbance is still linear in 40% solutions. For absorbance measurements at 280 and 310 nm, which are those made on agarose-suspension electrophoresis fractions (7), linearity between absorbance and Tween 20 concentration exists for all samples in which absorbance lies within the measurable range of the instrument. The extinction coefficients for a 1% solution in water and 0.1 M Tris-HCl, pH 8.0, were as follows: E&,,~~~ = 0.245, E:?,,,~~~ = 0.125. Agarose-Suspension Electrophoresis of Tween 20: Spectrophotometric Measurements
The migration of a 20% aqueous solution of Tween 20 in detergentfree agarose-suspension electrophoresis is illustrated in Fig. 2. It is evident from Fig. 2 that the changes in absorbance observed correspond to those originated by the presence of detergent and that these changes are absent in fractions that do not contain detergent. The detergent moves toward the cathode, i.e., in the opposite direction of the displacement of the solubilized membrane proteins (6). The extent of its migration was shown to be related linearly to the duration of the run in nine runs, each with a duration between 2 and 22 hr.
174
DRESDNER
AND CID-DRESDNER 0.9
a
B
=
2.0 6 .n i5 21.0
0.7
C
: a : :
0.5 ,20
40
60 Fraction
Ll
20
a
number
r.1, 360 Wavelength
500 ktrd
FIG. 2. Agarose-suspension electrophoresis of Tween 20. The sample contained 300 ~1 of aqueous 20% Tween 20 and the agarose from a 0.5ml suspension obtained by centrifugation. The agarose suspension contained 0.17% agarose and was prepared in the buffer used in the run (0.1 M Tris-HCI, pH 8.0). Samples migrating to the cathode move to the left of the starting point. (A) Representative run with a duration of 7 hr. (B) Control from the experiment shown in A. The sample contained water instead of detergent. The value of the differential absorbance of an aliquot of the same agarose suspension used in the run but kept in the cold room was 0.49. (C) Absorption spectrum of a sample obtained from the run shown in A by pooling fractions 14 and 15. The absorbance of the sample was measured against the three reference solutions indicated by different symbols. These were: pooled fractions 9 and 10 (0 0), pooled fractions 24 and 25 (0 l ), and 0.1 M Tris-HCI, pH 8.0 (0 0).
Surface-tension Measurements of Tween 20 Solutions Surface-tension measurements were chosen as an independent way to test the migration of Tween 20 in agarose-suspension electrophoresis. The technique showed a sensitive response for detergent concentrations as low as 0.001%. The results are shown in Fig. 3 in which three different concentration ranges have been represented. The O-0.1% concentration range is obviously the most useful as it is the most discriminative. The concentration of detergent in an appropriately diluted sample can be calculated from the data of Fig. 3. Agarose-Suspension Electrophoresis of Tween 20: Surface-tension and Spectrophotometric Measurements Samples of Tween 20 with different concentrations were run in agarosesuspension electrophoresis columns prepared in the absence of detergent.
~~~l~
o.cm4 FIG.
0.012
a020 0.2 0.6 1.0 2 Concentration of Tween 20 (g/100 ml)
6
3. Surface tension of Tween 20 solutions prepared in 0.1 M Tris-HCl,
10
pH 8.0, at 23°C.
175
REMOVALOFNEUTRALDETERGENT
At the end of the run, the fractions collected were centrifuged to remove the agarose. The differential absorbance, AZSO- ASl0, was measured before removing the agarose in some cases and afterward in other cases. To obtain surface-tension values corresponding to the O-0.1% concentration range of the detergent, most of the fractions from the “detergent peak” were diluted in 0.1 M Tris-HCl, pH 8.0, prior to measurement. A representative result is shown in Fig. 4. This experiment illustrates the wide range of detergent concentrations in the sample that can be handled with the method. The concentrations of detergent commonly used in agarose-suspension electrophoresis of solubilized A. laidlawii protein are, however, 5-8 times lower than those used in the experiment shown in Fig. 4, and the absorbance values that are usually measured are accordingly lower. Agarose-Suspension Electrophoresis of Human Serum Albumin Horse Heart Cytochrome c Mixed with Tween 20
and
The method was tested on pure proteins that had no previous contact with detergent. The proteins were mixed with Tween 20, and the mixtures were run in agarose-suspension electrophoresis. The results of these runs are shown in Fig. 5. In the case of cytochrome c (Fig. 5B), both the protein and the detergent migrate towards the cathode. Surface-tension measurements were performed on aliquots of these proteins, dialyzed against 0.1 M Tris-HCl, pH 8.0, before adding detergent and after agarose-suspension electrophoresis. The surface activity of a sample collected from agarose-suspension electrophoresis was compared with that of an aliquot of detergent-free protein of equal concentration. Concentration was determined spectrophotometrically. The results of these measurements are shown in Table 1. It is apparent from these results that detergent and protein separate as expected. Agarose-Suspension Electrophoresis Membrane Proteins
of A. laidlawii
A fraction of partially purified solubilized A. laidlawii membrane proteins was tested. The fraction was obtained by agarose-suspension electrophoresis of a sample of Tween 20 supernatant from solubilized A. laidlawii membranes, the run being performed in the presence of 1% Tween 20 (6,9). The fraction was composed mainly of the so called fraction T, which contains the proteins T, (flavoprotein) and Tar,; but, it also contained other proteins and some lipids. This is a representative fraction of Tween 20-soluble A. laidfawii membrane proteins, as T, and Tdb alone represent about 50% of the Tween 20-soluble membrane proteins and 30% of the total membrane proteins. The fraction was concentrated 10 times before agarose-suspension electrophoresis. After
176
DRESDNER
0.8 -
AND CID-DRESDNER
-8
' b-a
A
'280310 Conc.Tween20-6 S.Start
3 + ‘Ei -4
20
Fraction
40
h 3 z c ii
.-g + 0
60
number
FIG. 4. Agarose-suspension electrophoresis of Tween 20. The sample of Tween 20 was prepared in 0.1 M Tris-HCl, pH 8.0, and mixed with the agarose that had been obtained by centrifuging an aliquot of the 0.1% suspension used to fill up the electrophoresis column. It contained 1 ml of 20% Tween 20 and agarose from 1 ml of suspension. Voltage: 800 V; duration of the run: 12 hr. At the end of the run, the fractions were pooled in groups of two and centrifuged to remove the agarose; the differential absorbance, A 280- Asl0, was measured on the supernatant. The concentration of Tween 20 was obtained from surface-tension measurements performed in groups of four pooled fractions.
concentration, the detergent concentration in the sample was estimated as 9%. In Fig. 6A are shown the results obtained from the detergent-free agarose-suspension electrophoresis of a sample of solubilized A. faidfawii membrane proteins. This run was conducted in parallel with the one shown in Fig. 4 and the results of the surface-tension measurements were expressed as the concentration of Tween 20 that would cause a change in the surface tension of water equal to that observed. The results show that there is a maximum that migrates to the cathode which corresponds to the detergent peak. There is another maximum in the region where the lipids migrate in agarose-suspension electrophoresis in either the presence (6) or absence of detergent (10). There is next a region with several larger peaks originating from the presence of the proteins. In the fractions collected from this region, there is a surface tension that varies between 52 and 58 dyne/cm. These values are in good agreement with similar values obtained in surface-tension measurements of other proteins (11) at similar concentrations. Comparison of the protein composition of the samples described in the preceding experiment, before and after agarose-suspension electrophoresis, was made by means of analytical polyacrylamide gel elec-
A
I77
REMOVALOFNEUTRALDETERGENT
1.5 P 0 c?
.’ 0
0.5
0
f
$
1.0
20
z
-
LO
60 Fraction
20
10
60
number
FIG. 5. Separation of Tween 20 from human serum albumin and horse heart cytochrome c by agarose-suspension electrophoresis. The agarose suspension contained 0.19% agarose and was prepared in the buffer used in the run (0.1 M Tris-HCI, pH 8.0). The position of the electrodes with respect to the electrophoresis column is indicated at the sides of the diagrams by positive and negative signs. Note the difference in polarity in both runs. The position of the sample at the beginning is indicated by S. (A) Human serum albumin: the sample contained 400 ~1 of a protein-detergent mixture (2% serum albumin, 6% Tween 20) and agarose which had been separated by centrifugation of 500 ~1 of suspension. Voltage: 800 V; current: 21 mA; duration of the run: 14 hr. The horizontal line indicates the A,,, - A3r,, value of the agarose suspension. (B) Horse heart cytochrome c. The sample contained 400 ~1 of a mixture of protein and detergent (3% cytochrome c, 6% Tween 20) and agarose as indicated in A. Voltage: 600 V; current: 16 mA; duration of the run: 19 hr. After the absorbance measurements of the fractions, those fractions that comprised the dark areas were pooled; the agarose was removed, the supematant was diluted and dialysed against 0.1 M Tris-HC1, pH 8.0, and the surface tension was measured (see Table 1).
trophoresis. The results (Fig. 6B) show that no protein components were removed from the mixture of solubilized membrane components during agarose-suspension electrophoresis, and, therefore, all the proteins were completely recovered from the fractions of the protein peaks. Dialysis of the Samples Fractions other than those of the detergent peaks of the different agarose-suspension electrophoresis runs of Tween 20 showed the presence of a very low surface activity (66-67 dyne/cm). The origin of this activity was not determined. Dialysis of the fraction in 0.1 M Tris-HCl for 48-72 hr at 2°C completely removed this residual activity. Those fractions containing membrane proteins showed an identical decrease in activity after dialysis; the surface tension measured after dialysis was, therefore, that of the proteins alone. DISCUSSION
The separation of Tween 20 from the solubilized membrane proteins of A. laidlad by agarose-suspension electrophoresis in a detergentfree buffer is based upon the neutral character of the detergent, the
178
DRESDNER
AND CID-DRESDNER
A 310 A,,,A-A “Cont. Tween 20
20 A
10
Fraction
60
number
FIG. 6. Agarose-suspension electrophoresis of solubilized A. luidlawii membrane proteins. (A) The sample was prepared as described in the legend to Fig. 4. It contained 300 ~1 of solution of membrane proteins. Voltage: 800 V; current: 24 mA; duration of the run: 18 hr. At the end of the run, the differential absorbance AZeO- Aal0 of each fraction was measured. Next, the fractions were pooled in groups of two each and centrifuged; the supematant was collected and diluted with 1.5 ml of 0.1 M Tris-HCI, pH 8.0, and the surface tension was measured. The differential absorbance of the agarose suspension is indicated by line b. (B) Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of A. laidlawii membrane proteins before (left) and after (right) the removal of detergent. For the designation of the bands see Ref. (9). The samples were run on the same gel but at distant places. The corresponding slabs were cut out and placed side by side as shown in the figure. TABLE
1
SURFACE-TENSION MEASUREMENTS OF PROTEIN SOLUTIONS COLLECTED AGAROSE~USPENSION ELECTROPHORESIS OF MIXTURES OE PROTEIN AND TWEEN 2W”
AFTER
Surface tension (dyne/cm) Protein Human serum albumin Horse heart cytochrome c
Concentration (gihter) 2.6 0.7
o The samples were dialyzed against 0.1 M Tris-HCI, * Illustrated in Fig. 5. c Removal by agarose suspension electrophoresis.
Before mixing with detergent
After removal of detergent’
58 69
58 67
pH 8.0, prior to measurement.
REMOVAL
OF NEUTRAL
DETERGENT
179
0 Fig. 6 (continued)
charged nature of the proteins, and the stability of the proteins in a detergent-free medium. The two methods (absorbance and surfacetension measurement) used in the present work to detect the presence of detergent in fractions collected from agarose-suspension electrophoresis of samples containing only detergent, provide consistent results. The two methods are further supported by measurements of fractions from runs on samples containing both detergent and proteins. Thus, agarose-suspension electrophoresis represents an efficient means for the bulk removal of Tween 20 from solubilized membrane proteins. Of the methods used to determine the concentration of Tween 20, the surface-tension method showed greater sensitivity than the spectrophoto-
180
DRESDNER
AND CID-DRESDNER
metric. Concentrations of Tween 20 as low as 0.001% can be determined by this method. Tween 20 shows a slow migration to the cathode at pH 8.0. In the case of several polyoxyethylene derivatives of lauryl and tridecyl alcohols, it has been postulated that charging of the molecules could arise from hydronium ion formation by the hydrogen-bonded water molecules (12). The results obtained in the present work with Tween 20 agree with this interpretation, although the possibility also exists that migration in agarose-suspension electrophoresis could be due to electroendosmosis. The slow migration of the detergent towards the cathode determines that, in fact, the separation, depends on the protein mobility, other factors being constant. With 500~~1 samples containing detergent concentrations lower than IO%, this method separates the detergent from proteins satisfactorily with a mobility similar to that of T,, or TJb (6), serum albumin, or cytochrome c under the conditions described here. If the concentration of the detergent is about 10% or higher, the use of a 0.19 instead of a 0.17% agarose suspension is advisable as it will confer more stability to the sample when placed in the column. It is obvious that separation of the detergent from the proteins will be possible only if both are stable in a detergent-free solution. This is particularly critical for membrane proteins. In the case of the A. faidfawii membrane proteins used in the present work, it has been found that they remain in solution at 2°C in the buffer used for electrophoresis for a period of at least 1 month. Concentration of the proteins, however, leads to precipitate formation. Since the principles involved in this method are not restricted to the system described here, therefore, it seems plausible that other neutral detergents can be similarly removed from nearly any protein, membranebound or not. It should also be possible to remove charged detergents, provided their mobilities differ sufficiently from those of the proteins of interest. In addition, the same principle can be applied using other types of electrophoretic techniques. The present technique might be used on a preparative scale. There are available, at present, large columns (13) that allow the use of 8 ml of a solution containing IO- 1.5 mg/ml of protein; this is 15 times larger than the capacity of the method described here and 20-30 times larger than that of preparative polyacrylamide gel electrophoresis (4). ACKNOWLEDGMENTS The authors want to express their gratitude to Professor Stellan Hjerten who provided all the facilities to do this work and to Dr. Karl-Erik Johansson and Mrs. Itja Blomqvist for teaching the agarose-suspension electrophoresis technique. During the time this work was being done, G.D. was the recipient of a fellowship from the Swedish International
REMOVAL
OF NEUTRAL
DETERGENT
181
Development Authority and the Swedish Institute. This work was supported by the von Kantzow Foundation, the Swedish Natural Science Research Council, and the Wallenberg Foundation. The support of these institutions is gratefully acknowledged.
REFERENCES 1. Razin, S. (1972) Biochim. Eiophys. Acta 265, 241-296. 2. Becher, P. (1967) in Nonionic Surfactants (Schick. M. J.. ed.), 1st ed., p. 48, Marcel Dekker, New York. 3. Rigg, M. W., and Liu, F. W. J. (1953) J. Amer. Oil Chem. Sot. 30, 14- 17. 4. Johansson, K.-E., Blomqvist, I.. and Hjerttn, S. (1975)JBioI. Chem. 250,2463-2469. 5. Gaylor, I. L., and Delwicke. C. V. (1968)Anal. Biochem. 28, 361-368. 6. Johansson, K.-E. (1973) Prot. Biol. Fluids 21, 151- 156. 7. Hjerten, S., Hoglund, S., and Ruttkay-Nedecky, G. (1970) Acta Virol 14, 89-101. 8. Lundahl, P., and Liljas, L. (1975) Anal. Eiochem. 65, 50-59. 9. Hjerten, S., and Johansson, K.-E. (1972) Biochim. Biophys. Acta 288, 312-325. 10. Dresdner. G., and Cid-Dresdner, H.. unpublished observations. 11. St. Johnston, J. H. (1927)Biochem. J. 21, 1314-1328. 12. Becher, P. (1962) 1. Colloid Sci. 17, 325-333. 13. Hjerten, S. (1963) J. Chromatog. 12, 510-526.
BIOCHEMISTRY
The Removal Solubilized
78, 171- 1st (1977)
of the Neutral Detergent Tween 20 from Membrane Proteins of Acholeplasma laidlawii and Other Proteins by Agarose-suspension Electrophoresis
GEORGE DRESDNER’AND HILDA CID-DRESDNER~ Membrane Group. Institute of Biochemistry, Biomedical Center, University Box 576, S-751 23 Uppsala, Sweden, and ZStructure Group, Wallenberg Uppsala University, Box 562, S-751 22 Uppsala, Sweden
of Uppsala, Laboratory,
Received June 29. 1976; accepted October 21. 1976 The neutral detergent Tween 20 can be detected in fractions from agarosesuspension electrophoresis by spectrophotometry and surface-tension measurements. A concentration of Tween 20 as low as 0.001% can be detected by surface-tension measurements. In runs performed at pH 8.0 in 0.1 M Tris-HCI, the detergent remains near the starting point. Using these conditions, the detergent can be removed from membrane proteins of Acholeplasma laidlawii solubilized by Tween 20 or from proteins such as human seturn albumin and horse heart cytochrome c to which detergent has been added. The method can be used on a IS-mg preparative scale.
When a neutral detergent is used for the separation and purification of membrane proteins, its removal from the purified fractions is often difficult (l), particularly when the sample has previously been concentrated. For instance, it cannot be removed by dialysis due to its large micellar size and its low critical micelle concentration which determines that a small difference in the concentration of free detergent will exist on both sides of the dialysis membrane (2-4). Successful removal of neutral detergents from proteins has been achieved by gel filtration (5) or by preparative polyacrylamide gel electrophoresis (6). In the latter case, the method has been used to remove the neutral detergent Tween 20 from purified membrane proteins of Acholeplasma laidlawii. But, with this technique, it is difficult to handle amounts of membrane proteins on a preparative scale (6,4). In the present work, agarose-suspension electrophoresis is shown to be a convenient technique for the removal of the neutral detergent Tween 20 from the solubilized membrane proteins of A. faidlnwii and from other proteins as well. r Present address: Biophysics Department, Arrhenius Laboratory. Stockholm University. S-104 05 Stockholm, Sweden.
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN Mw)3-2697
172
DRESDNER
AND CID-DRESDNER
MATERIALS
AND METHODS
Chemicals. All chemicals were reagent grade. Agarose powder was purchased from Bio-Rad Laboratories; Tween 20, quality SD, was purchased from Atlas Chemie, GmbH, Essen, Germany. Human serum albumin was from AB Kabi, Stockholm, Sweden. Horse heart cytochrome c from Sigma Chemical Co., St. Louis, Missouri was a gift of Dr. Per Lundahl . Agarose-suspension electrophoresis. Details of the technique have already been published (7,4). The agarose suspension was prepared in 0.1 M Tris-HCl, pH 8.0, and it contained 0.02% sodium azide. Spectrophotometric measurements. Absorbance measurements were performed in a PMQ III Zeiss spectrophotometer using quartz cuvettes with a l-cm pathlength. The absorbance of agarose-suspension electrophoresis fractions was measured in a PMQ II Zeiss spectrophotometer as already described (7). Absorption spectra were measured with a Model 25 Beckman recording spectrophotometer. Surface-tension measurements. A capillary-rise apparatus with a graduated capillary tube was used for surface-tension measurements. The apparatus was calibrated with distilled, filtered, and deionized water. Measurements were performed at 23°C. Analytical polyacrylamide gel electrophoresis. Electrophoresis in the presence of 0.02 M sodium dodecyl sulfate and 0.1 M Tris-HCI (pH 8.0) was performed on 0.6 x 10 x lO-cm gel slabs at room temperature. Details of the apparatus and technique have already been published (8). Gels with the following composition were used: T = 6%, C = 5% (8). With a voltage gradient of 10 V/cm, separation was achieved within 90 min. The gels were stained with Coomassie brilliant blue R 250. Sample concentration. In some cases, the samples were concentrated with a B- 1.5 Minicon miniconcentrator prior to analytical polyacrylamide gel electrophoresis. RESULTS
Two different techniques were used to detect the presence of detergent in fractions obtained from agarose-suspension electrophoresis. These were spectrophotometric and surface-tension measurements. The techniques were first standardized by performing measurements on Tween 20 solutions. Spectrophotometric
Measurements of Tween 20 Solutions
The absorption spectrum of Tween 20 is shown in Fig. 1A. gent solution was prepared in 0.1 M Tris-HCl, pH 8.0. Figure that part of the spectrum that is used in practice with the concentration commonly present in mixtures of solubilized
The deter1B shows Tween 20 membrane
REMOVAL
A
DETERGENT
.
173
C
.
12 .I
,” 2.0 c 2 0’ 2
8 LL
OF NEUTRAL
3
/
.
.I//
1.0
a
.I/
Kl
300
LOO
Wavelength
250
350
(nm)
10
30
Tween 20 concentrationp/.)
FIG. 1. Spectrophotometric measurements of Tween 20 solutions in 0.1 M Tris-HCI, pH 8.0. (A) Absorption spectrum of a 1% solution. (B) Absorption spectrum of a 3% solution; the figure shows that part of the spectrum that is used in practice. (C) Relationship between absorbance and concentration of a Tween 20 solution at different wavelengths: (I) 280. (2) 310, and (3) 350 nm.
proteins of A. laidlawii. Even when the spectrum shows a monotonous increase of absorbance towards shorter wavelengths, the detergent can be clearly distinguished from the background in fractions from agarosesuspension electrophoresis. Figure 1C shows that Tween 20 solutions follow Bourger- Lambert- Beer’s law within the range of concentrations examined. The maximal concentration studied was 40%. Its absorbance at 280 nm is larger than the maximally measurable absorbance of the instrument. But, measurements at lower wavelengths show that the relationship between Tween 20 concentration and absorbance is still linear in 40% solutions. For absorbance measurements at 280 and 310 nm, which are those made on agarose-suspension electrophoresis fractions (7), linearity between absorbance and Tween 20 concentration exists for all samples in which absorbance lies within the measurable range of the instrument. The extinction coefficients for a 1% solution in water and 0.1 M Tris-HCl, pH 8.0, were as follows: E&,,~~~ = 0.245, E:?,,,~~~ = 0.125. Agarose-Suspension Electrophoresis of Tween 20: Spectrophotometric Measurements
The migration of a 20% aqueous solution of Tween 20 in detergentfree agarose-suspension electrophoresis is illustrated in Fig. 2. It is evident from Fig. 2 that the changes in absorbance observed correspond to those originated by the presence of detergent and that these changes are absent in fractions that do not contain detergent. The detergent moves toward the cathode, i.e., in the opposite direction of the displacement of the solubilized membrane proteins (6). The extent of its migration was shown to be related linearly to the duration of the run in nine runs, each with a duration between 2 and 22 hr.
174
DRESDNER
AND CID-DRESDNER 0.9
a
B
=
2.0 6 .n i5 21.0
0.7
C
: a : :
0.5 ,20
40
60 Fraction
Ll
20
a
number
r.1, 360 Wavelength
500 ktrd
FIG. 2. Agarose-suspension electrophoresis of Tween 20. The sample contained 300 ~1 of aqueous 20% Tween 20 and the agarose from a 0.5ml suspension obtained by centrifugation. The agarose suspension contained 0.17% agarose and was prepared in the buffer used in the run (0.1 M Tris-HCI, pH 8.0). Samples migrating to the cathode move to the left of the starting point. (A) Representative run with a duration of 7 hr. (B) Control from the experiment shown in A. The sample contained water instead of detergent. The value of the differential absorbance of an aliquot of the same agarose suspension used in the run but kept in the cold room was 0.49. (C) Absorption spectrum of a sample obtained from the run shown in A by pooling fractions 14 and 15. The absorbance of the sample was measured against the three reference solutions indicated by different symbols. These were: pooled fractions 9 and 10 (0 0), pooled fractions 24 and 25 (0 l ), and 0.1 M Tris-HCI, pH 8.0 (0 0).
Surface-tension Measurements of Tween 20 Solutions Surface-tension measurements were chosen as an independent way to test the migration of Tween 20 in agarose-suspension electrophoresis. The technique showed a sensitive response for detergent concentrations as low as 0.001%. The results are shown in Fig. 3 in which three different concentration ranges have been represented. The O-0.1% concentration range is obviously the most useful as it is the most discriminative. The concentration of detergent in an appropriately diluted sample can be calculated from the data of Fig. 3. Agarose-Suspension Electrophoresis of Tween 20: Surface-tension and Spectrophotometric Measurements Samples of Tween 20 with different concentrations were run in agarosesuspension electrophoresis columns prepared in the absence of detergent.
~~~l~
o.cm4 FIG.
0.012
a020 0.2 0.6 1.0 2 Concentration of Tween 20 (g/100 ml)
6
3. Surface tension of Tween 20 solutions prepared in 0.1 M Tris-HCl,
10
pH 8.0, at 23°C.
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At the end of the run, the fractions collected were centrifuged to remove the agarose. The differential absorbance, AZSO- ASl0, was measured before removing the agarose in some cases and afterward in other cases. To obtain surface-tension values corresponding to the O-0.1% concentration range of the detergent, most of the fractions from the “detergent peak” were diluted in 0.1 M Tris-HCl, pH 8.0, prior to measurement. A representative result is shown in Fig. 4. This experiment illustrates the wide range of detergent concentrations in the sample that can be handled with the method. The concentrations of detergent commonly used in agarose-suspension electrophoresis of solubilized A. laidlawii protein are, however, 5-8 times lower than those used in the experiment shown in Fig. 4, and the absorbance values that are usually measured are accordingly lower. Agarose-Suspension Electrophoresis of Human Serum Albumin Horse Heart Cytochrome c Mixed with Tween 20
and
The method was tested on pure proteins that had no previous contact with detergent. The proteins were mixed with Tween 20, and the mixtures were run in agarose-suspension electrophoresis. The results of these runs are shown in Fig. 5. In the case of cytochrome c (Fig. 5B), both the protein and the detergent migrate towards the cathode. Surface-tension measurements were performed on aliquots of these proteins, dialyzed against 0.1 M Tris-HCl, pH 8.0, before adding detergent and after agarose-suspension electrophoresis. The surface activity of a sample collected from agarose-suspension electrophoresis was compared with that of an aliquot of detergent-free protein of equal concentration. Concentration was determined spectrophotometrically. The results of these measurements are shown in Table 1. It is apparent from these results that detergent and protein separate as expected. Agarose-Suspension Electrophoresis Membrane Proteins
of A. laidlawii
A fraction of partially purified solubilized A. laidlawii membrane proteins was tested. The fraction was obtained by agarose-suspension electrophoresis of a sample of Tween 20 supernatant from solubilized A. laidlawii membranes, the run being performed in the presence of 1% Tween 20 (6,9). The fraction was composed mainly of the so called fraction T, which contains the proteins T, (flavoprotein) and Tar,; but, it also contained other proteins and some lipids. This is a representative fraction of Tween 20-soluble A. laidfawii membrane proteins, as T, and Tdb alone represent about 50% of the Tween 20-soluble membrane proteins and 30% of the total membrane proteins. The fraction was concentrated 10 times before agarose-suspension electrophoresis. After
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'280310 Conc.Tween20-6 S.Start
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h 3 z c ii
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FIG. 4. Agarose-suspension electrophoresis of Tween 20. The sample of Tween 20 was prepared in 0.1 M Tris-HCl, pH 8.0, and mixed with the agarose that had been obtained by centrifuging an aliquot of the 0.1% suspension used to fill up the electrophoresis column. It contained 1 ml of 20% Tween 20 and agarose from 1 ml of suspension. Voltage: 800 V; duration of the run: 12 hr. At the end of the run, the fractions were pooled in groups of two and centrifuged to remove the agarose; the differential absorbance, A 280- Asl0, was measured on the supernatant. The concentration of Tween 20 was obtained from surface-tension measurements performed in groups of four pooled fractions.
concentration, the detergent concentration in the sample was estimated as 9%. In Fig. 6A are shown the results obtained from the detergent-free agarose-suspension electrophoresis of a sample of solubilized A. faidfawii membrane proteins. This run was conducted in parallel with the one shown in Fig. 4 and the results of the surface-tension measurements were expressed as the concentration of Tween 20 that would cause a change in the surface tension of water equal to that observed. The results show that there is a maximum that migrates to the cathode which corresponds to the detergent peak. There is another maximum in the region where the lipids migrate in agarose-suspension electrophoresis in either the presence (6) or absence of detergent (10). There is next a region with several larger peaks originating from the presence of the proteins. In the fractions collected from this region, there is a surface tension that varies between 52 and 58 dyne/cm. These values are in good agreement with similar values obtained in surface-tension measurements of other proteins (11) at similar concentrations. Comparison of the protein composition of the samples described in the preceding experiment, before and after agarose-suspension electrophoresis, was made by means of analytical polyacrylamide gel elec-
A
I77
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1.5 P 0 c?
.’ 0
0.5
0
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$
1.0
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z
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LO
60 Fraction
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10
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FIG. 5. Separation of Tween 20 from human serum albumin and horse heart cytochrome c by agarose-suspension electrophoresis. The agarose suspension contained 0.19% agarose and was prepared in the buffer used in the run (0.1 M Tris-HCI, pH 8.0). The position of the electrodes with respect to the electrophoresis column is indicated at the sides of the diagrams by positive and negative signs. Note the difference in polarity in both runs. The position of the sample at the beginning is indicated by S. (A) Human serum albumin: the sample contained 400 ~1 of a protein-detergent mixture (2% serum albumin, 6% Tween 20) and agarose which had been separated by centrifugation of 500 ~1 of suspension. Voltage: 800 V; current: 21 mA; duration of the run: 14 hr. The horizontal line indicates the A,,, - A3r,, value of the agarose suspension. (B) Horse heart cytochrome c. The sample contained 400 ~1 of a mixture of protein and detergent (3% cytochrome c, 6% Tween 20) and agarose as indicated in A. Voltage: 600 V; current: 16 mA; duration of the run: 19 hr. After the absorbance measurements of the fractions, those fractions that comprised the dark areas were pooled; the agarose was removed, the supematant was diluted and dialysed against 0.1 M Tris-HC1, pH 8.0, and the surface tension was measured (see Table 1).
trophoresis. The results (Fig. 6B) show that no protein components were removed from the mixture of solubilized membrane components during agarose-suspension electrophoresis, and, therefore, all the proteins were completely recovered from the fractions of the protein peaks. Dialysis of the Samples Fractions other than those of the detergent peaks of the different agarose-suspension electrophoresis runs of Tween 20 showed the presence of a very low surface activity (66-67 dyne/cm). The origin of this activity was not determined. Dialysis of the fraction in 0.1 M Tris-HCl for 48-72 hr at 2°C completely removed this residual activity. Those fractions containing membrane proteins showed an identical decrease in activity after dialysis; the surface tension measured after dialysis was, therefore, that of the proteins alone. DISCUSSION
The separation of Tween 20 from the solubilized membrane proteins of A. laidlad by agarose-suspension electrophoresis in a detergentfree buffer is based upon the neutral character of the detergent, the
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FIG. 6. Agarose-suspension electrophoresis of solubilized A. luidlawii membrane proteins. (A) The sample was prepared as described in the legend to Fig. 4. It contained 300 ~1 of solution of membrane proteins. Voltage: 800 V; current: 24 mA; duration of the run: 18 hr. At the end of the run, the differential absorbance AZeO- Aal0 of each fraction was measured. Next, the fractions were pooled in groups of two each and centrifuged; the supematant was collected and diluted with 1.5 ml of 0.1 M Tris-HCI, pH 8.0, and the surface tension was measured. The differential absorbance of the agarose suspension is indicated by line b. (B) Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of A. laidlawii membrane proteins before (left) and after (right) the removal of detergent. For the designation of the bands see Ref. (9). The samples were run on the same gel but at distant places. The corresponding slabs were cut out and placed side by side as shown in the figure. TABLE
1
SURFACE-TENSION MEASUREMENTS OF PROTEIN SOLUTIONS COLLECTED AGAROSE~USPENSION ELECTROPHORESIS OF MIXTURES OE PROTEIN AND TWEEN 2W”
AFTER
Surface tension (dyne/cm) Protein Human serum albumin Horse heart cytochrome c
Concentration (gihter) 2.6 0.7
o The samples were dialyzed against 0.1 M Tris-HCI, * Illustrated in Fig. 5. c Removal by agarose suspension electrophoresis.
Before mixing with detergent
After removal of detergent’
58 69
58 67
pH 8.0, prior to measurement.
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0 Fig. 6 (continued)
charged nature of the proteins, and the stability of the proteins in a detergent-free medium. The two methods (absorbance and surfacetension measurement) used in the present work to detect the presence of detergent in fractions collected from agarose-suspension electrophoresis of samples containing only detergent, provide consistent results. The two methods are further supported by measurements of fractions from runs on samples containing both detergent and proteins. Thus, agarose-suspension electrophoresis represents an efficient means for the bulk removal of Tween 20 from solubilized membrane proteins. Of the methods used to determine the concentration of Tween 20, the surface-tension method showed greater sensitivity than the spectrophoto-
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metric. Concentrations of Tween 20 as low as 0.001% can be determined by this method. Tween 20 shows a slow migration to the cathode at pH 8.0. In the case of several polyoxyethylene derivatives of lauryl and tridecyl alcohols, it has been postulated that charging of the molecules could arise from hydronium ion formation by the hydrogen-bonded water molecules (12). The results obtained in the present work with Tween 20 agree with this interpretation, although the possibility also exists that migration in agarose-suspension electrophoresis could be due to electroendosmosis. The slow migration of the detergent towards the cathode determines that, in fact, the separation, depends on the protein mobility, other factors being constant. With 500~~1 samples containing detergent concentrations lower than IO%, this method separates the detergent from proteins satisfactorily with a mobility similar to that of T,, or TJb (6), serum albumin, or cytochrome c under the conditions described here. If the concentration of the detergent is about 10% or higher, the use of a 0.19 instead of a 0.17% agarose suspension is advisable as it will confer more stability to the sample when placed in the column. It is obvious that separation of the detergent from the proteins will be possible only if both are stable in a detergent-free solution. This is particularly critical for membrane proteins. In the case of the A. faidfawii membrane proteins used in the present work, it has been found that they remain in solution at 2°C in the buffer used for electrophoresis for a period of at least 1 month. Concentration of the proteins, however, leads to precipitate formation. Since the principles involved in this method are not restricted to the system described here, therefore, it seems plausible that other neutral detergents can be similarly removed from nearly any protein, membranebound or not. It should also be possible to remove charged detergents, provided their mobilities differ sufficiently from those of the proteins of interest. In addition, the same principle can be applied using other types of electrophoretic techniques. The present technique might be used on a preparative scale. There are available, at present, large columns (13) that allow the use of 8 ml of a solution containing IO- 1.5 mg/ml of protein; this is 15 times larger than the capacity of the method described here and 20-30 times larger than that of preparative polyacrylamide gel electrophoresis (4). ACKNOWLEDGMENTS The authors want to express their gratitude to Professor Stellan Hjerten who provided all the facilities to do this work and to Dr. Karl-Erik Johansson and Mrs. Itja Blomqvist for teaching the agarose-suspension electrophoresis technique. During the time this work was being done, G.D. was the recipient of a fellowship from the Swedish International
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Development Authority and the Swedish Institute. This work was supported by the von Kantzow Foundation, the Swedish Natural Science Research Council, and the Wallenberg Foundation. The support of these institutions is gratefully acknowledged.
REFERENCES 1. Razin, S. (1972) Biochim. Eiophys. Acta 265, 241-296. 2. Becher, P. (1967) in Nonionic Surfactants (Schick. M. J.. ed.), 1st ed., p. 48, Marcel Dekker, New York. 3. Rigg, M. W., and Liu, F. W. J. (1953) J. Amer. Oil Chem. Sot. 30, 14- 17. 4. Johansson, K.-E., Blomqvist, I.. and Hjerttn, S. (1975)JBioI. Chem. 250,2463-2469. 5. Gaylor, I. L., and Delwicke. C. V. (1968)Anal. Biochem. 28, 361-368. 6. Johansson, K.-E. (1973) Prot. Biol. Fluids 21, 151- 156. 7. Hjerten, S., Hoglund, S., and Ruttkay-Nedecky, G. (1970) Acta Virol 14, 89-101. 8. Lundahl, P., and Liljas, L. (1975) Anal. Eiochem. 65, 50-59. 9. Hjerten, S., and Johansson, K.-E. (1972) Biochim. Biophys. Acta 288, 312-325. 10. Dresdner. G., and Cid-Dresdner, H.. unpublished observations. 11. St. Johnston, J. H. (1927)Biochem. J. 21, 1314-1328. 12. Becher, P. (1962) 1. Colloid Sci. 17, 325-333. 13. Hjerten, S. (1963) J. Chromatog. 12, 510-526.