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Affinity chromatography of rat plasma albumin on bromosulfophthalein-glutathione conjugate covalently linked to Sepharose 4-B
ANALYTICAL
BIOCHEMISTRY
92. 290-293
(1979)
Affinity Chromatography of Rat Plasma Albumin Bromosulfophthalein-Glutathione Conjugate Covalently Linked to Sepharose 4-B
In a recent publication (I), we described a matrix for the affinity chromatography of glutathione S-transferase. The matrix was bromosulfophthalein-glutathione conjugate covalently linked to Sepharose 4-B. It was observed in this publication that this matrix could be used for the preparation of rat plasma albumin and we now wish to describe this application in greater detail and to compare the properties of this matrix with those of matrixes described by other workers.
0.01 M sodium phosphate, pH 7.4. The sample was loaded on to a column (0.6 x 2.5 cm) of the affinity matrix which had previously been equilibrated with this buffer. The sample was washed through the column with buffer A until the extinction at 280 nm had fallen to zero. A 20-m] gradient of sodium chloride (O-3.0 M. buffered at pH 7.4 with 0.01~ sodium phosphate) was then applied. A flow rate of 12 mlihr was employed for all operations which were carried out at room temperature. Fractions of I ml were collected and their protein content was monitored by measuring their extinction at 280 nm in 2-mm pathlength spectrophotometer cells. When appropriate. protein-containing samples were exhaustively dialyzed against distilled water prior to analytical electrophoresis. In one series of experiments, solutions of sulfobromophthalein (1 or 10 mM. 10 ml) were used to elute bound protein. Because of the high extinction of this dye under uv. it was not possible to monitor the eluted protein spectrophotometrically. In these instances. the protein content of the eluted fractions was examined qualitatively by analytical electrophoresis as described below. To determine the capacity of the gel. the columns were saturated with protein by applying 25 ml of diluted plasma. The columns were washed as described above and bound protein was eluted with 2 M NaCl solution. Quantitative protein determinations were
MATERIALS
Sulfobromophthalein and L-glutathione were purchased from Koch-Light Laboratories Ltd. and CNBr-activated Sepharose 4-B from Pharmacia. Blood was taken from ether-anaesthetized rats of both sexes into a heparinized syringe from the posterior vena cava. It was centrifuged for 20 min at lOOO,r: at room temperature. The supernatant was decanted and stored at -20°C until required. METHODS Sywtlwsis of rrffiwity support. Sulfobromophthalein-glutathione conjugate was synthesized and immobilized on Sepharose 4-B as described previously (1). Af$nity (,hroIIlrrtoXrcrph~ of pl~~.smr~ nlh~lrrli~. In developing the technique, the following procedure was adopted. Plasma (0.2 ml) was diluted to 1 ml with buffer A. 0003.2697/79/020290-04$02,00/O Copynght All right\
$1 1979 by Academx Pre\r. Inc. uf reproductwn m any form rewvcd.
on
290
AFFINITY
CHROMATOGRAPHY
Fraction
FIG. I. Chromatography linked to Sepharose 4-B.
RESULTS
AND DISCUSSION
The record of a small-scale preparation, utilizing a sodium chloride gradient, is shown in Fig. 1. The bulk of the protein (65% as determined from the extinction at ’ Abbreviation
used:
number
of rat plasma proteins on sulfobromophthalein-glutathione Experimental details are described in the text.
made by the method of Lowry rt ~1. (4) using bovine serum albumin as standard. For routine preparations of albumin, columns of up to IO ml (1.5 x 5 cm) were employed. Plasma (2 ml) was diluted fivefold with 0.1 M sodium chloride solution buffered at pH 7.4 with 0.01 M sodium phosphate. The same solution was used to wash the columns, and elution of bound proteins was effected with buffered 2 M sodium chloride which eluted the bound protein within 2 column volumes. The eluted protein was exhaustively dialyzed against distilled water and then lyophilized. Electrophorrsis. Disc electrophoresis in polyacrylamide gels was carried out by standard methods (5,6). Protein zones were localized by staining with Coomassie blue R (7). Stained gels were scanned at 620 nm with an IX0 scanning densitometer. Sodium dodecylsulfate (SDS)’ electrophoresis in 10% polyacrylamide gels was carried out as described by Weber and Osborne (8).
SDS.
sodium
dodecyl
sulfate.
291
OF ALBUMIN
conjugate
covalently
280 nm of the eluted fractions) passes straight through the column and is rapidly cleared from it. The remainder is eluted as a single peak at NaCl concentrations of between 0.5 and 1.5 M. Samples of protein from the breakthrough peak and of protein eluted from the column were examined by electrophoresis in polyacrylamide gel. In Fig. 2, the results of scanning gels, in which protein samples from (a) the peak of the unretained fractions and (b) the peak of the fractions eluted with salt had been subjected to electrophoresis, are shown. It is clear that the retained material consists almost entirely of a single protein (>98%) the mobility of which is that of albumin. The efficiency of extraction of albumin is shown in that the unretained material contains virtually no albumin at all. It was observed that the first two fractions in the peak of salt-eluted protein contained traces (~7%) of contaminating proteins. The addition of 0.1 M NaCl to the running buffer prevented the binding of these proteins and albumin obtained in routine preparations, in which the higher ionic strength buffer was used and in which elution was achieved with 2 M NaCl, was of a purity almost comparable with that obtained with gradient elutions (see Fig. 3a). Electrophoresis in polyacrylamide gel revealed the
292
CLARK
presence of a minor, slowly migrating contaminant in albumin prepared by stepwise elution. Densitometry on the stained gels suggested that this contaminant accounted for no more than 3% of the protein present. On electrophoresis in SDS, three contaminating proteins were detectable (see Fig. 3b). Two well-defined bands of mobility slightly greater than that of albumin were seen, as well as a diffuse band of still greater mobility. These three bands accounted for approximately 4% of the total stained protein. A similar pattern was seen if the chromatography was carried out at 4°C. If any of these contaminants are products of partial proteolysis of albumin, then it seems likely that the proteolysis occurs prior to the chromatography step. Preliminary experiments show that the albumins from mouse and human plasma are also adsorbed to the matrix and may be eluted with high concentrations of salt. That the adsorption of albumin was specific was shown by the immediate elution (over 2-3 fractions) of albumin by solutions of bromosulfophthalein at concentrations as
” 0
FIG.
gels of of rat fraction gradient eluted protein, elution
2 L 01stance
6 1 mugrated
2
L
6
(cm)
2. Analytical electrophoresis in polyacrylamide proteins obtained after affinity chromatography plasma. (a) Unretained protein (sample from 3. Fig. 1). (b) Protein eked with a salt (fraction 31. Fig. 1). (c) Retained protein, with 10 mM sulfobromophthalein. (d) Retained eluted with 10 mM sulfobromophthalein after of albumin with 2 M NaCl solution.
AND
WONG 2[
I 1t
n
:: 2
0
L0
/,uI, 2
4 Distance
L, 6 migrated
0
2
4
6
(cm)
FIG. 3. Analytical electrophoresis of rat plasma albumin prepared by stepwise elution from the affinity column, (a) Electrophoresis of 100 wg of albumin in a standard polyacrylamide gel system(5.6). (b) Electrophoresis of 100 pg of albumin in 0.1% SDS in polyacrylamide gel (8). Staining and densitometry were carried out as described under Methods.
low as 1 mM. This dye was not used routinely because of its high extinction under uv and because of the difficulty of removing it from the albumin. Furthermore, the protein eluted contained two major contaminating proteins (Fig. 2~). These proteins were not eluted by sodium chloride, and elution with salt followed by elution with bromosulfophthalein yielded these contaminating proteins accompanied by only traces of albumin (Fig. 2d). This experiment demonstrates the relative specificity and the high efficiency of salt elution, only a trace of albumin is left after salt elution. The identity of these proteins is unknown. Tests for glutathione S-transferase activity, which, if present, might be expected to be eluted with sulfobromophthalein (1) were negative. The capacity of the affinity matrix for rat plasma albumin was found to be 7.5 mg of albumin/ml of gel. This was adequate for our requirements. Yields of 50 mg of homogeneous albumin were routinely obtained during preparative runs. The extent of substitution on the gel was 2.2 pmoli
AFFINITY
CHROMATOGRAPHY
ml of gel (1). Higher degrees of substitution might be expected from freshly activated Sepharose 4-B but it seems unlikely that the present method would yield a matrix with the capacity reported for immobilized Cibacron Blue (2). There may, however, be advantages to the present method. Albumin solutions obtained by the method in Ref. (2) were described as being bright yellow in color. possibly indicating the presence of bound bilirubin. Albumin obtained by the present method was quite colorless. Since bilirubin and dyes of the phthalein type are known to compete for the same high-affinity sites on albumin [e.g., see (9)], the use of such a dye as an immobilized ligand would be expecteld to yield a bilirubin-free product. If the color of the albumin obtained by Travis et al. (2) was indeed due to bilirubin then it seems likely that the two methods exploit diffierent modes of binding to the protein. It has been suggested (2) that Cibacron Blue may bind at a nucleotide binding site (IO). The metlhod of Peters et ~1. (3), in which immobilized fatty acids were employed as an affinity .matrix, may take advantage of a different mode of binding again. It seems unlikely that the bromosulfophthalein-binding site is involved since fatty acids do not compete with bilirubin for high-affinity binding (I 1) and hence should not compete with phthalein dyes (9). It seems likely therefore that, in addition to its preparative applications, the affinity matrix reported here, in combination with those described by other workers (2,3)
OF
ALBUMIN
293
may prove a useful tool in studying and distinguishing the various modes of ligand binding to serum albumins (12). ACKNOWLEDGMENTS This work has been supported in part by grants from the Internal Research Committee of Victoria University and from the Lottery Profits Distribution Committee. S.T.W. is grateful to the Wellington Medical Research Foundation for financial support. The authors also wish to acknowledge the technical assistance of Ms. H. Parkin.
REFERENCES 1. Clark. A. G., Letoa, M., and Wong, S. T. (1977) Lif, Sri. 20, 141- 148. 2. Travis. J., Bowen. J., Tewksbury. D., Johnson, D., and Pannel, R. (1976) Bioc~hen~. J. 157, 301-306. 3. Peters. T.. Taniuchi. H.. and Anlinsen, C. (1973) J. Biol. C’het~~. 248, 2447-2451. 4. Lowry. 0. H., Rosebrough. N. J., Farr. A. L.. and Randall. R. J. (1951) J. Biol. Chrm. 193, 256-275. 5. Davis, B. T. (1964) Am. N. Y. Acud. Sci. 121. 404-427. 6. Ornstein, L. (1964) Ann. N. Y. Awd. .Sc,i. 121. 321-349. 7. Vesterberg, 0. (1972) Biochir,~. Bioplzys. Acrcr 257, I l-19. 8. Weber. K.. and Osborne, M. (1969).1. Biol. Chrm. 244, 4406-4412. 9. Hertz, H. (1975) Srcrrzd. J. C/in. Lob. Inwst. 35, 545-559. IO. Thomson. S. T., Cass. K. H., and Stellwagen. E. (1975) Fed. Proc,. Fed. Amcjr. SIIC,. Exp. Biol. 34, 567. 11. Jacobsen. J.. Thiessen. H., and Brodersen. R. ( 1972) Bioc,hrm. J. 126, 7P. 12. Nichol. L. W., Ogston, A. G.. Winzor. D. J.. and Sawyer, W. H. (1974) Biochm. J. 143, 435% 443.
BIOCHEMISTRY
92. 290-293
(1979)
Affinity Chromatography of Rat Plasma Albumin Bromosulfophthalein-Glutathione Conjugate Covalently Linked to Sepharose 4-B
In a recent publication (I), we described a matrix for the affinity chromatography of glutathione S-transferase. The matrix was bromosulfophthalein-glutathione conjugate covalently linked to Sepharose 4-B. It was observed in this publication that this matrix could be used for the preparation of rat plasma albumin and we now wish to describe this application in greater detail and to compare the properties of this matrix with those of matrixes described by other workers.
0.01 M sodium phosphate, pH 7.4. The sample was loaded on to a column (0.6 x 2.5 cm) of the affinity matrix which had previously been equilibrated with this buffer. The sample was washed through the column with buffer A until the extinction at 280 nm had fallen to zero. A 20-m] gradient of sodium chloride (O-3.0 M. buffered at pH 7.4 with 0.01~ sodium phosphate) was then applied. A flow rate of 12 mlihr was employed for all operations which were carried out at room temperature. Fractions of I ml were collected and their protein content was monitored by measuring their extinction at 280 nm in 2-mm pathlength spectrophotometer cells. When appropriate. protein-containing samples were exhaustively dialyzed against distilled water prior to analytical electrophoresis. In one series of experiments, solutions of sulfobromophthalein (1 or 10 mM. 10 ml) were used to elute bound protein. Because of the high extinction of this dye under uv. it was not possible to monitor the eluted protein spectrophotometrically. In these instances. the protein content of the eluted fractions was examined qualitatively by analytical electrophoresis as described below. To determine the capacity of the gel. the columns were saturated with protein by applying 25 ml of diluted plasma. The columns were washed as described above and bound protein was eluted with 2 M NaCl solution. Quantitative protein determinations were
MATERIALS
Sulfobromophthalein and L-glutathione were purchased from Koch-Light Laboratories Ltd. and CNBr-activated Sepharose 4-B from Pharmacia. Blood was taken from ether-anaesthetized rats of both sexes into a heparinized syringe from the posterior vena cava. It was centrifuged for 20 min at lOOO,r: at room temperature. The supernatant was decanted and stored at -20°C until required. METHODS Sywtlwsis of rrffiwity support. Sulfobromophthalein-glutathione conjugate was synthesized and immobilized on Sepharose 4-B as described previously (1). Af$nity (,hroIIlrrtoXrcrph~ of pl~~.smr~ nlh~lrrli~. In developing the technique, the following procedure was adopted. Plasma (0.2 ml) was diluted to 1 ml with buffer A. 0003.2697/79/020290-04$02,00/O Copynght All right\
$1 1979 by Academx Pre\r. Inc. uf reproductwn m any form rewvcd.
on
290
AFFINITY
CHROMATOGRAPHY
Fraction
FIG. I. Chromatography linked to Sepharose 4-B.
RESULTS
AND DISCUSSION
The record of a small-scale preparation, utilizing a sodium chloride gradient, is shown in Fig. 1. The bulk of the protein (65% as determined from the extinction at ’ Abbreviation
used:
number
of rat plasma proteins on sulfobromophthalein-glutathione Experimental details are described in the text.
made by the method of Lowry rt ~1. (4) using bovine serum albumin as standard. For routine preparations of albumin, columns of up to IO ml (1.5 x 5 cm) were employed. Plasma (2 ml) was diluted fivefold with 0.1 M sodium chloride solution buffered at pH 7.4 with 0.01 M sodium phosphate. The same solution was used to wash the columns, and elution of bound proteins was effected with buffered 2 M sodium chloride which eluted the bound protein within 2 column volumes. The eluted protein was exhaustively dialyzed against distilled water and then lyophilized. Electrophorrsis. Disc electrophoresis in polyacrylamide gels was carried out by standard methods (5,6). Protein zones were localized by staining with Coomassie blue R (7). Stained gels were scanned at 620 nm with an IX0 scanning densitometer. Sodium dodecylsulfate (SDS)’ electrophoresis in 10% polyacrylamide gels was carried out as described by Weber and Osborne (8).
SDS.
sodium
dodecyl
sulfate.
291
OF ALBUMIN
conjugate
covalently
280 nm of the eluted fractions) passes straight through the column and is rapidly cleared from it. The remainder is eluted as a single peak at NaCl concentrations of between 0.5 and 1.5 M. Samples of protein from the breakthrough peak and of protein eluted from the column were examined by electrophoresis in polyacrylamide gel. In Fig. 2, the results of scanning gels, in which protein samples from (a) the peak of the unretained fractions and (b) the peak of the fractions eluted with salt had been subjected to electrophoresis, are shown. It is clear that the retained material consists almost entirely of a single protein (>98%) the mobility of which is that of albumin. The efficiency of extraction of albumin is shown in that the unretained material contains virtually no albumin at all. It was observed that the first two fractions in the peak of salt-eluted protein contained traces (~7%) of contaminating proteins. The addition of 0.1 M NaCl to the running buffer prevented the binding of these proteins and albumin obtained in routine preparations, in which the higher ionic strength buffer was used and in which elution was achieved with 2 M NaCl, was of a purity almost comparable with that obtained with gradient elutions (see Fig. 3a). Electrophoresis in polyacrylamide gel revealed the
292
CLARK
presence of a minor, slowly migrating contaminant in albumin prepared by stepwise elution. Densitometry on the stained gels suggested that this contaminant accounted for no more than 3% of the protein present. On electrophoresis in SDS, three contaminating proteins were detectable (see Fig. 3b). Two well-defined bands of mobility slightly greater than that of albumin were seen, as well as a diffuse band of still greater mobility. These three bands accounted for approximately 4% of the total stained protein. A similar pattern was seen if the chromatography was carried out at 4°C. If any of these contaminants are products of partial proteolysis of albumin, then it seems likely that the proteolysis occurs prior to the chromatography step. Preliminary experiments show that the albumins from mouse and human plasma are also adsorbed to the matrix and may be eluted with high concentrations of salt. That the adsorption of albumin was specific was shown by the immediate elution (over 2-3 fractions) of albumin by solutions of bromosulfophthalein at concentrations as
” 0
FIG.
gels of of rat fraction gradient eluted protein, elution
2 L 01stance
6 1 mugrated
2
L
6
(cm)
2. Analytical electrophoresis in polyacrylamide proteins obtained after affinity chromatography plasma. (a) Unretained protein (sample from 3. Fig. 1). (b) Protein eked with a salt (fraction 31. Fig. 1). (c) Retained protein, with 10 mM sulfobromophthalein. (d) Retained eluted with 10 mM sulfobromophthalein after of albumin with 2 M NaCl solution.
AND
WONG 2[
I 1t
n
:: 2
0
L0
/,uI, 2
4 Distance
L, 6 migrated
0
2
4
6
(cm)
FIG. 3. Analytical electrophoresis of rat plasma albumin prepared by stepwise elution from the affinity column, (a) Electrophoresis of 100 wg of albumin in a standard polyacrylamide gel system(5.6). (b) Electrophoresis of 100 pg of albumin in 0.1% SDS in polyacrylamide gel (8). Staining and densitometry were carried out as described under Methods.
low as 1 mM. This dye was not used routinely because of its high extinction under uv and because of the difficulty of removing it from the albumin. Furthermore, the protein eluted contained two major contaminating proteins (Fig. 2~). These proteins were not eluted by sodium chloride, and elution with salt followed by elution with bromosulfophthalein yielded these contaminating proteins accompanied by only traces of albumin (Fig. 2d). This experiment demonstrates the relative specificity and the high efficiency of salt elution, only a trace of albumin is left after salt elution. The identity of these proteins is unknown. Tests for glutathione S-transferase activity, which, if present, might be expected to be eluted with sulfobromophthalein (1) were negative. The capacity of the affinity matrix for rat plasma albumin was found to be 7.5 mg of albumin/ml of gel. This was adequate for our requirements. Yields of 50 mg of homogeneous albumin were routinely obtained during preparative runs. The extent of substitution on the gel was 2.2 pmoli
AFFINITY
CHROMATOGRAPHY
ml of gel (1). Higher degrees of substitution might be expected from freshly activated Sepharose 4-B but it seems unlikely that the present method would yield a matrix with the capacity reported for immobilized Cibacron Blue (2). There may, however, be advantages to the present method. Albumin solutions obtained by the method in Ref. (2) were described as being bright yellow in color. possibly indicating the presence of bound bilirubin. Albumin obtained by the present method was quite colorless. Since bilirubin and dyes of the phthalein type are known to compete for the same high-affinity sites on albumin [e.g., see (9)], the use of such a dye as an immobilized ligand would be expecteld to yield a bilirubin-free product. If the color of the albumin obtained by Travis et al. (2) was indeed due to bilirubin then it seems likely that the two methods exploit diffierent modes of binding to the protein. It has been suggested (2) that Cibacron Blue may bind at a nucleotide binding site (IO). The metlhod of Peters et ~1. (3), in which immobilized fatty acids were employed as an affinity .matrix, may take advantage of a different mode of binding again. It seems unlikely that the bromosulfophthalein-binding site is involved since fatty acids do not compete with bilirubin for high-affinity binding (I 1) and hence should not compete with phthalein dyes (9). It seems likely therefore that, in addition to its preparative applications, the affinity matrix reported here, in combination with those described by other workers (2,3)
OF
ALBUMIN
293
may prove a useful tool in studying and distinguishing the various modes of ligand binding to serum albumins (12). ACKNOWLEDGMENTS This work has been supported in part by grants from the Internal Research Committee of Victoria University and from the Lottery Profits Distribution Committee. S.T.W. is grateful to the Wellington Medical Research Foundation for financial support. The authors also wish to acknowledge the technical assistance of Ms. H. Parkin.
REFERENCES 1. Clark. A. G., Letoa, M., and Wong, S. T. (1977) Lif, Sri. 20, 141- 148. 2. Travis. J., Bowen. J., Tewksbury. D., Johnson, D., and Pannel, R. (1976) Bioc~hen~. J. 157, 301-306. 3. Peters. T.. Taniuchi. H.. and Anlinsen, C. (1973) J. Biol. C’het~~. 248, 2447-2451. 4. Lowry. 0. H., Rosebrough. N. J., Farr. A. L.. and Randall. R. J. (1951) J. Biol. Chrm. 193, 256-275. 5. Davis, B. T. (1964) Am. N. Y. Acud. Sci. 121. 404-427. 6. Ornstein, L. (1964) Ann. N. Y. Awd. .Sc,i. 121. 321-349. 7. Vesterberg, 0. (1972) Biochir,~. Bioplzys. Acrcr 257, I l-19. 8. Weber. K.. and Osborne, M. (1969).1. Biol. Chrm. 244, 4406-4412. 9. Hertz, H. (1975) Srcrrzd. J. C/in. Lob. Inwst. 35, 545-559. IO. Thomson. S. T., Cass. K. H., and Stellwagen. E. (1975) Fed. Proc,. Fed. Amcjr. SIIC,. Exp. Biol. 34, 567. 11. Jacobsen. J.. Thiessen. H., and Brodersen. R. ( 1972) Bioc,hrm. J. 126, 7P. 12. Nichol. L. W., Ogston, A. G.. Winzor. D. J.. and Sawyer, W. H. (1974) Biochm. J. 143, 435% 443.