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Isolation of 3,4-dihydroxyphenylalanine-containing proteins using boronate affinity chromatography
ANALYTICAL
BIOCHEMISTRY
159, 187- 190 ( 1986)
Isolation of 3,4-Dihydroxyphenylalanine-Containing Using Boronate Affinity Chromatography CLIFFORD J. HAWKINS,*
Proteins
MARTIN F. LAWN,? DAVID L. PARRY,* AND IAN L. Ross?
*Department of Chemistry, and TDepartment of Biochemistry, University of Queensland, St Lucia, Australia 4067 Received April 29, 1986 A rapid procedure for the isolation of 3,4dihydroxyphenylalanine-containing proteins has been developed in which the protein is selectively bound to a m-phenylboronate agarose column, and eluted with 1.OM ammonium acetate, pH 3.0. The method is based on the affinity of boronates for diols including catechol. The chromatography is carried out in the absence of oxygen to prevent oxidation of the catechol. Other proteins are eluted beforehand with 0.25 M ammonium acetate, pH 8.5, or for glycoproteins with a Tris buffer containing 0.2 M sorbitol, pH 8.5. 0 1986 Academic
KEY
Press, Inc.
WORDS:
protein purification; structural proteins; DOPA-proteins; iron-binding
proteins;
ascidians.
3,4-Dihydroxyphenylalanine (DOPA)’ has been found as an integral component of proteins that are involved in the formation of sclerotized structures in invertebrates ( 1,2), and in the adhesion of sessile marine organisms to their substrates (3,4). It is postulated that these processes involve the oxidation of the catechol group to quinone and the attack of the quinonoid ring by free amine groups to cross-link proteins (4). This facile oxidation, cross-linking, and precipitation interferes with the isolation of pure DOPA-protein from neutral or basic solutions. The tendency of the proteins to precipitate with anionic compounds such as sodium dodecylsulfate (SDS), and to adsorb to chromatographic media also interferes with the application of standard techniques such as gel filtration, ion-exchange, and polyacrylamide gel electrophoresis (3). An iron-binding DOPA-protein, ferreascidin, has been isolated from the blood cells of the stolidobranch ascidian, Pyura stolonifea ’ Abbreviations used: DOPA, 3,4dihydroxyphenylalamine; SDS, sodium dodecylsulfate; PAGE, polyacrylamide gel electrophoresis; CTAB, cetyltrimethylammonium bromide.
(unpublished results). A method of isolation and purification of the protein has been developed based on the known formation of strong but reversible complexes between boronate and catechols, which have the advantage of inhibiting the oxidation of the catechols (5). The method utilizes a m-phenylboronate agarose column, and provides a simple, rapid method for both the detection and the purification of DOPA-containing proteins. Immobilized phenylboronate columns have been used previously for the separation of catecholic amino acids and catecholamines (6). MATERIALS
AND METHODS
Most reagents were obtained commercially: Matrix Gel PBA-10 and PBA-30 m-phenylboronate agarose from Amicon; GlycoGe1.B from Pierce; cytochrome c, ovalbumin, bovine serum albumin, tranferrin, L-tyrosine, and LDOPA from Sigma; ovine hemoglobin from Seravac; lysozyme from Boehringer-Mannheim. All polyacrylamide gel electrophoresis and buffer components were analytical grade. Isolation of ferreascidin. The protein was isolated from the blood cells of P. stolonifera which had been collected from local seashores 187
0003-2697186 $3.00 CopyrigJ~t 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved
188
HAWKINS
and kept in aquaria until required. Blood was collected on ice and the yellow cells were pelleted at 3000g for 5 min at 4°C. The protein was extracted from the cells by vigorous resuspension in cold 0.25 M ammonium acetate, pH 4.5, for 10 min. Following centrifugation the extract was stored at -70°C. Phenylboronate afinity chromatography. A Matrix Gel PBA-10 column (bed volume 2.5 ml) was equilibrated at 25°C with buffer A (0.25 M ammonium acetate, pH 8.5) at a flow rate of 1.0 ml/min. Stock protein solutions with the exception of ferreascidin were prepared at 10 mg ml-’ in buffer A. The L-tyrosine solution was prepared at a concentration of 0.5 mM in buffer A made 5% with ethanol. To prevent the rapid oxidation of DOPA that occurs at alkaline pH, stock solutions of LDOPA (0.5 mM) were prepared in deoxygenated buffer A. The deoxygenation was achieved by heating the solution to near-boiling, then plunging the solution into an icebath while sparging vigorously with nitrogen until the solution had cooled to room temperature. The pH of the buffer was checked and readjusted to pH 8.5 with ammonia if necessary. The deoxygenated solutions were stored under nitrogen in a VAC D&Lab glove box. Prior to chromatography, ferreascidin solutions (&so - 0.8) were dialyzed exhaustively under nitrogen against deoxygenated buffer A. For the chromatography, 500-~1 aliquots of stock solutions were applied to the column, with the exception of ferreascidin for which a 3-ml sample was used. To remove adsorbed material other than the DOPA-protein, the column was washed with 15 ml of buffer A. To elute more tightly bound protein 15 ml of buffer B (0.1 M Tris-HCl, 0.2 M sorbitol, 10 mM EDTA, pH 8.5) was applied, followed by 5 ml of buffer A to remove sorbitol. Elution of DOPA-containing protein was achieved with 15 ml of 0.1 M ammonium acetate, pH 3.0. Fractions (1.5 ml) were collected throughout, and uv-visible spectra recorded using a Hewlett-Packard 8450A spectrophotometer. All chromatography was carried out
ET
AL.
at 25 “C, and under an atmosphere of nitrogen for L-DOPA and ferreascidin. Similar experiments with ferreascidin were carried out with Matrix Gel PBA-30 and GlycoGe1.B columns. DOPA assays. The presence of DOPA in ferreascidin was assayed spectrophotometritally using a modified procedure of the borate difference method described by Waite and Tanzer (2), and by HPLC after acid hydrolysis (6 M HCl, 108°C 48 h) using postcolumn OPA derivatization and fluorescence detection. The former method relies on a large increase in the absorbance of DOPA upon the formation of the borate complex at pH 8.0, with maximum difference at 294 nm. Gel electrophoresis. Polyacrylamide gel electrophoresis (PAGE) was performed at pH 4.56 in the presence of the cationic detergent, cetyltrimethylammonium bromide (CTAB), using the stacking system described by Mocz and Balint (7), and using riboflavin as initiator. SDS-PAGE was also carried out using the method of Laemmli (8) with 0.1 M borate in all buffers to prevent oxidation of DOPA. RESULTS
The DOPA-containing protein, ferreascidin, was found to bind tightly to Matrix Gel PBA10 during application and washing with buffer A. The protein also remained on the gel during attempted elution with sorbitol buffer (buffer B) which is the usual displacement ligand employed for this type of matrix. Elution offerreascidin was achieved with ammonium acetate, pH 3.0. A number of other proteins and amino acids were chromatographed under similar conditions. The results in Fig. 1 describe elution profiles for bovine serum albumin, ferreascidin, and DOPA. Cytochrome c, ovalbumin, transferrin, ovine hemoglobin, lysozyme, and tyrosine as well as bovine serum albumin showed no affinity for the gel and were eluted from the column by buffer A. The catecholic amino acid L-DOPA remained bound to the column under washing conditions but was eluted with sorbitol buffer. Fer-
BORONATE
AFFINITY
189
METHOD
band from GlycoGel.B, while 30% acetic acid was necessary in the case of PBA-30. Glycoproteins also bound more tightly to these two Sorbitol AC&ate materials. I I The presence of DOPA in ferreascidin was confirmed by HPLC (Fig. 2) and by spectrophotometric analyses. The purity of the ferreascidin used in these experiments was determined by CTAB and SDS-PAGE. The results using CTAB demonstrate that most of the protein appears as a single band of approximately 12,000 Da (Fig. 3). Although ferreascidin precipitates with SDS, sufficient material remains in solution to allow convenFIG. I. Elution profiles of bovine serum albumin (0). tional SDS-PAGE which also shows a single DOPA (A), and ferreascidin (m) from PBA-10 phenylboband at approximately the same molecular ronate agarose. Elution was performed in three stages; washing with Buffer A, Buffer B (sorbitol), and pH 3 ac- weight. etate, respectively. DISCUSSION
reascidin remains bound to the column under conditions where all other proteins and amino acids have been eluted. It was also found that the presence of CTAB allowed slow elution at pH 5.0. Matrix Gel PBA-30 and GlycoGe1.B were found to bind the DOPA-protein more tightly than Matrix Gel PBA-10: a slightly lower pH (pH 2.5) was necessary to effect a sharp elution
The applications of immobilized boronate ligand affinity chromatography to the purification of carbohydrates, nucleic acids, and glycoproteins have been described previously (9). The reversible hydroxyl exchange between the boronate group and 1,2-cis-diols to form a five-membered ring provides an ideal mechanism for the affinity chromatography of catechols, and is the basis for the method of iso-
F c
ln,ectio” I
L. I
20
40 Retention
60 time
60
(min)
FIG. 2. HPLC profile of amino acid hydrolysate of ferreascidin. DOPA is present at approximately four residues per hundred.
190
HAWKINS
1234
-
SI”
FIG. 3. CTAB-PAGE of ferreascidin. Lanes 2 and 3 contain ferreascidin (20 and 10 pg, respectively). Lanes 1 and 4 contain commercial protein standards: soybean trypsin inhibitor (20,100 Da), carbonic anhydrase (30,000 Da), ovalbumin (43,000 Da), bovine serum albumin (67,000 Da), phosphorylase b (94,000 Da). The distortion observed appears to be due to the less efficient binding of CTAB to proteins than for SDS.
ET AL.
The observation that the cationic detergent CTAB allows elution of the ferreascidin at pH 5 rather than pH 3 would be valuable for proteins that are particularly sensitive to acid hydrolysis. The cationic detergent also facilitates the electrophoresis of ferreascidin whereas SDS precipitates the protein precluding conventional SDS-PAGE. This has been observed for some other DOPA-proteins (3). The CTAB-PAGE has the added advantage of a low pH which inhibits the oxidation of DGPA. ACKNOWLEDGMENTS This work was supported by funds from the University of Queensland, and a Marine Sciences and Technologies Grant (C.J.H.).
REFERENCES
lating DOPA-proteins described in this paper. The method is simple, rapid, and highly specific. The Matrix Gel PBA- 10 material has a low ligand concentration (lo- 15 pmol-’ ml) and is especially designed for tightly binding macromolecules (9). It was found to be the most suitable matrix for the separation of the DGPA-proteins from other proteins, including glycoproteins. The catechol groups in ferreascidin bind the protein to the gel more tightly than glycoproteins, and the presence of multiple catechol groups, with other secondary interactions between the protein and the derivatized gel, make the binding of the DOPAprotein to the gel more stable than for simple catechols. Greater ligand concentrations as in Matrix Gel PBA-30 (30-50 pmol-’ ml) reduced the specificity and required the elution of the DOPA-protein at lower pH.
1. Hunt, S. (1970) Polysaccharide-Protein Complexes in Invertebrates, Academic Press, New York. 2. Waite, J. H., and Benedict, C. V. (1984) in Methods in Enzymology (Wold, F., and Moldave, K., eds.), Vol. 107, pp. 397-413, Academic Press,New York. 3. Waite, J. H., and Tamer, M. L. (1981) Science 212, 1038-1040. 4. Lindner, E., and Dooley, C. A. (1976) in Proc. 4th International Congress on Marine Corrosion and Fouling, pp. 333-344, Ant&es, Juan&s-Pins, France. 5. Steinberg, H. (1964) Organoboron Chemistry, Vol. I, Wiley, New York. 6. Hansson, C., Agrup, G., Rorsman, H., Rosengren, A. M., and Rosengren, E. (1978) J Chromatogr. 161,352-355. 7.
Mocz, G., and Bahnt, M. (1984) Anal. Biochem. 143,
8.
Laemmli, J. K. (1970) Nature (London) 227, 680-
9.
Amicon, (1981) Boronate Ligands in Biochemical Separations, Amicon Corp., Danvers Mass. and references therein.
283-292. 685.
BIOCHEMISTRY
159, 187- 190 ( 1986)
Isolation of 3,4-Dihydroxyphenylalanine-Containing Using Boronate Affinity Chromatography CLIFFORD J. HAWKINS,*
Proteins
MARTIN F. LAWN,? DAVID L. PARRY,* AND IAN L. Ross?
*Department of Chemistry, and TDepartment of Biochemistry, University of Queensland, St Lucia, Australia 4067 Received April 29, 1986 A rapid procedure for the isolation of 3,4dihydroxyphenylalanine-containing proteins has been developed in which the protein is selectively bound to a m-phenylboronate agarose column, and eluted with 1.OM ammonium acetate, pH 3.0. The method is based on the affinity of boronates for diols including catechol. The chromatography is carried out in the absence of oxygen to prevent oxidation of the catechol. Other proteins are eluted beforehand with 0.25 M ammonium acetate, pH 8.5, or for glycoproteins with a Tris buffer containing 0.2 M sorbitol, pH 8.5. 0 1986 Academic
KEY
Press, Inc.
WORDS:
protein purification; structural proteins; DOPA-proteins; iron-binding
proteins;
ascidians.
3,4-Dihydroxyphenylalanine (DOPA)’ has been found as an integral component of proteins that are involved in the formation of sclerotized structures in invertebrates ( 1,2), and in the adhesion of sessile marine organisms to their substrates (3,4). It is postulated that these processes involve the oxidation of the catechol group to quinone and the attack of the quinonoid ring by free amine groups to cross-link proteins (4). This facile oxidation, cross-linking, and precipitation interferes with the isolation of pure DOPA-protein from neutral or basic solutions. The tendency of the proteins to precipitate with anionic compounds such as sodium dodecylsulfate (SDS), and to adsorb to chromatographic media also interferes with the application of standard techniques such as gel filtration, ion-exchange, and polyacrylamide gel electrophoresis (3). An iron-binding DOPA-protein, ferreascidin, has been isolated from the blood cells of the stolidobranch ascidian, Pyura stolonifea ’ Abbreviations used: DOPA, 3,4dihydroxyphenylalamine; SDS, sodium dodecylsulfate; PAGE, polyacrylamide gel electrophoresis; CTAB, cetyltrimethylammonium bromide.
(unpublished results). A method of isolation and purification of the protein has been developed based on the known formation of strong but reversible complexes between boronate and catechols, which have the advantage of inhibiting the oxidation of the catechols (5). The method utilizes a m-phenylboronate agarose column, and provides a simple, rapid method for both the detection and the purification of DOPA-containing proteins. Immobilized phenylboronate columns have been used previously for the separation of catecholic amino acids and catecholamines (6). MATERIALS
AND METHODS
Most reagents were obtained commercially: Matrix Gel PBA-10 and PBA-30 m-phenylboronate agarose from Amicon; GlycoGe1.B from Pierce; cytochrome c, ovalbumin, bovine serum albumin, tranferrin, L-tyrosine, and LDOPA from Sigma; ovine hemoglobin from Seravac; lysozyme from Boehringer-Mannheim. All polyacrylamide gel electrophoresis and buffer components were analytical grade. Isolation of ferreascidin. The protein was isolated from the blood cells of P. stolonifera which had been collected from local seashores 187
0003-2697186 $3.00 CopyrigJ~t 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved
188
HAWKINS
and kept in aquaria until required. Blood was collected on ice and the yellow cells were pelleted at 3000g for 5 min at 4°C. The protein was extracted from the cells by vigorous resuspension in cold 0.25 M ammonium acetate, pH 4.5, for 10 min. Following centrifugation the extract was stored at -70°C. Phenylboronate afinity chromatography. A Matrix Gel PBA-10 column (bed volume 2.5 ml) was equilibrated at 25°C with buffer A (0.25 M ammonium acetate, pH 8.5) at a flow rate of 1.0 ml/min. Stock protein solutions with the exception of ferreascidin were prepared at 10 mg ml-’ in buffer A. The L-tyrosine solution was prepared at a concentration of 0.5 mM in buffer A made 5% with ethanol. To prevent the rapid oxidation of DOPA that occurs at alkaline pH, stock solutions of LDOPA (0.5 mM) were prepared in deoxygenated buffer A. The deoxygenation was achieved by heating the solution to near-boiling, then plunging the solution into an icebath while sparging vigorously with nitrogen until the solution had cooled to room temperature. The pH of the buffer was checked and readjusted to pH 8.5 with ammonia if necessary. The deoxygenated solutions were stored under nitrogen in a VAC D&Lab glove box. Prior to chromatography, ferreascidin solutions (&so - 0.8) were dialyzed exhaustively under nitrogen against deoxygenated buffer A. For the chromatography, 500-~1 aliquots of stock solutions were applied to the column, with the exception of ferreascidin for which a 3-ml sample was used. To remove adsorbed material other than the DOPA-protein, the column was washed with 15 ml of buffer A. To elute more tightly bound protein 15 ml of buffer B (0.1 M Tris-HCl, 0.2 M sorbitol, 10 mM EDTA, pH 8.5) was applied, followed by 5 ml of buffer A to remove sorbitol. Elution of DOPA-containing protein was achieved with 15 ml of 0.1 M ammonium acetate, pH 3.0. Fractions (1.5 ml) were collected throughout, and uv-visible spectra recorded using a Hewlett-Packard 8450A spectrophotometer. All chromatography was carried out
ET
AL.
at 25 “C, and under an atmosphere of nitrogen for L-DOPA and ferreascidin. Similar experiments with ferreascidin were carried out with Matrix Gel PBA-30 and GlycoGe1.B columns. DOPA assays. The presence of DOPA in ferreascidin was assayed spectrophotometritally using a modified procedure of the borate difference method described by Waite and Tanzer (2), and by HPLC after acid hydrolysis (6 M HCl, 108°C 48 h) using postcolumn OPA derivatization and fluorescence detection. The former method relies on a large increase in the absorbance of DOPA upon the formation of the borate complex at pH 8.0, with maximum difference at 294 nm. Gel electrophoresis. Polyacrylamide gel electrophoresis (PAGE) was performed at pH 4.56 in the presence of the cationic detergent, cetyltrimethylammonium bromide (CTAB), using the stacking system described by Mocz and Balint (7), and using riboflavin as initiator. SDS-PAGE was also carried out using the method of Laemmli (8) with 0.1 M borate in all buffers to prevent oxidation of DOPA. RESULTS
The DOPA-containing protein, ferreascidin, was found to bind tightly to Matrix Gel PBA10 during application and washing with buffer A. The protein also remained on the gel during attempted elution with sorbitol buffer (buffer B) which is the usual displacement ligand employed for this type of matrix. Elution offerreascidin was achieved with ammonium acetate, pH 3.0. A number of other proteins and amino acids were chromatographed under similar conditions. The results in Fig. 1 describe elution profiles for bovine serum albumin, ferreascidin, and DOPA. Cytochrome c, ovalbumin, transferrin, ovine hemoglobin, lysozyme, and tyrosine as well as bovine serum albumin showed no affinity for the gel and were eluted from the column by buffer A. The catecholic amino acid L-DOPA remained bound to the column under washing conditions but was eluted with sorbitol buffer. Fer-
BORONATE
AFFINITY
189
METHOD
band from GlycoGel.B, while 30% acetic acid was necessary in the case of PBA-30. Glycoproteins also bound more tightly to these two Sorbitol AC&ate materials. I I The presence of DOPA in ferreascidin was confirmed by HPLC (Fig. 2) and by spectrophotometric analyses. The purity of the ferreascidin used in these experiments was determined by CTAB and SDS-PAGE. The results using CTAB demonstrate that most of the protein appears as a single band of approximately 12,000 Da (Fig. 3). Although ferreascidin precipitates with SDS, sufficient material remains in solution to allow convenFIG. I. Elution profiles of bovine serum albumin (0). tional SDS-PAGE which also shows a single DOPA (A), and ferreascidin (m) from PBA-10 phenylboband at approximately the same molecular ronate agarose. Elution was performed in three stages; washing with Buffer A, Buffer B (sorbitol), and pH 3 ac- weight. etate, respectively. DISCUSSION
reascidin remains bound to the column under conditions where all other proteins and amino acids have been eluted. It was also found that the presence of CTAB allowed slow elution at pH 5.0. Matrix Gel PBA-30 and GlycoGe1.B were found to bind the DOPA-protein more tightly than Matrix Gel PBA-10: a slightly lower pH (pH 2.5) was necessary to effect a sharp elution
The applications of immobilized boronate ligand affinity chromatography to the purification of carbohydrates, nucleic acids, and glycoproteins have been described previously (9). The reversible hydroxyl exchange between the boronate group and 1,2-cis-diols to form a five-membered ring provides an ideal mechanism for the affinity chromatography of catechols, and is the basis for the method of iso-
F c
ln,ectio” I
L. I
20
40 Retention
60 time
60
(min)
FIG. 2. HPLC profile of amino acid hydrolysate of ferreascidin. DOPA is present at approximately four residues per hundred.
190
HAWKINS
1234
-
SI”
FIG. 3. CTAB-PAGE of ferreascidin. Lanes 2 and 3 contain ferreascidin (20 and 10 pg, respectively). Lanes 1 and 4 contain commercial protein standards: soybean trypsin inhibitor (20,100 Da), carbonic anhydrase (30,000 Da), ovalbumin (43,000 Da), bovine serum albumin (67,000 Da), phosphorylase b (94,000 Da). The distortion observed appears to be due to the less efficient binding of CTAB to proteins than for SDS.
ET AL.
The observation that the cationic detergent CTAB allows elution of the ferreascidin at pH 5 rather than pH 3 would be valuable for proteins that are particularly sensitive to acid hydrolysis. The cationic detergent also facilitates the electrophoresis of ferreascidin whereas SDS precipitates the protein precluding conventional SDS-PAGE. This has been observed for some other DOPA-proteins (3). The CTAB-PAGE has the added advantage of a low pH which inhibits the oxidation of DGPA. ACKNOWLEDGMENTS This work was supported by funds from the University of Queensland, and a Marine Sciences and Technologies Grant (C.J.H.).
REFERENCES
lating DOPA-proteins described in this paper. The method is simple, rapid, and highly specific. The Matrix Gel PBA- 10 material has a low ligand concentration (lo- 15 pmol-’ ml) and is especially designed for tightly binding macromolecules (9). It was found to be the most suitable matrix for the separation of the DGPA-proteins from other proteins, including glycoproteins. The catechol groups in ferreascidin bind the protein to the gel more tightly than glycoproteins, and the presence of multiple catechol groups, with other secondary interactions between the protein and the derivatized gel, make the binding of the DOPAprotein to the gel more stable than for simple catechols. Greater ligand concentrations as in Matrix Gel PBA-30 (30-50 pmol-’ ml) reduced the specificity and required the elution of the DOPA-protein at lower pH.
1. Hunt, S. (1970) Polysaccharide-Protein Complexes in Invertebrates, Academic Press, New York. 2. Waite, J. H., and Benedict, C. V. (1984) in Methods in Enzymology (Wold, F., and Moldave, K., eds.), Vol. 107, pp. 397-413, Academic Press,New York. 3. Waite, J. H., and Tamer, M. L. (1981) Science 212, 1038-1040. 4. Lindner, E., and Dooley, C. A. (1976) in Proc. 4th International Congress on Marine Corrosion and Fouling, pp. 333-344, Ant&es, Juan&s-Pins, France. 5. Steinberg, H. (1964) Organoboron Chemistry, Vol. I, Wiley, New York. 6. Hansson, C., Agrup, G., Rorsman, H., Rosengren, A. M., and Rosengren, E. (1978) J Chromatogr. 161,352-355. 7.
Mocz, G., and Bahnt, M. (1984) Anal. Biochem. 143,
8.
Laemmli, J. K. (1970) Nature (London) 227, 680-
9.
Amicon, (1981) Boronate Ligands in Biochemical Separations, Amicon Corp., Danvers Mass. and references therein.
283-292. 685.