ANALYTICAL BIOCHEMISTRY 70, 542-546 (1976)
Removal of Ampholines by Hollow Fiber Dialysis: Application to Dextransucrase M I C H A E L F . C A L L A H A M , W I L L I A M E . POE, AND JAMES R . H E I T Z
Mississippi State University, Mississippi State, Mississippi 39762 Received June 2, 1975; accepted September 2, 1975 Ampholines must he removed from proteins that have been purified using isoelectric focusing chromatography before hydrolysis since ninhydrin positive fragments result. Hollow fiber dialysis was shown to be a fast and efficient method for the removal of ampholines from dextransucrase after isoelectric focusing. The purified protein was then subjected to amino acid analysis.
In enzyme purifications utilizing isoelectric focusing chromatography as one of the methods of purification, it is necessary to remove the ampholines preparatory to determining the amino acid composition of the enzyme. The earliest theories concerning the ease of removal of ampholines by membrane dialysis (1) have been modified by an awareness that the removal by this means may be difficult (2,3). Studies using ampholines alone suggest that the ampholine polymers may be too large to pass the dialysis membrane. Since other techniques of ampholine removal from purified protein, such as gel filtration, electrophoresis, or salt precipitation, may not be applicable in all cases, another alternative method would be desirable. This report is concerned with the use of hollow fiber dialysis as a means of removal of ampholines from dextransucrase, an enzyme that has been purified from bacterial sources using isoelectric focusing chromatography as the terminal step (4,5). This also allows the determination of the amino acid composition of dextransucrase. MATERIALS AND METHODS A 2.5% (v/v) solution o f p H 3 - l0 ampholines (LKB Producter, Sweden) in deionized water was prepared and divided into three parts. Solution I was immediately frozen. Solution II (50 ml) was placed in dialysis tubing, which had been pretreated according to a published procedure (6). Dialysis was carried out for 24 hr against 500 ml of 0.05 M sodium phosphate buffer, pH 6.5, with four buffer changes and constant stirring at 4°C. At the end of this treatment, Solution II was removed and frozen. Solution III was placed in a Beaker Dialyzer (Bio-Rad, Richmond, California) containing cellulose Bio Fiber 50 with a 5000 MW cut off, and the solution was dialyzed. The ampholine solution was placed outside the fibers and 542 Copyright© 1976by AcademicPress, Inc. All rightsof reproductionin any form reserved.
543
REMOVAL OF AMPHOLINES
deionized water, at an approximate flow rate of 200 ml/min, was allowed to pass through the fibers. The dialysis procedure was carried out for 1 hr with constant stirring of the ampholine mixture. This ampholine Solution III was removed and frozen. The three samples were placed in a Virtis Freeze Drying apparatus and lyophilyzed to dryness. Duplicate samples of the three ampholine solutions were hydrolyzed according to a published procedure (7). Dextransucrase was purified according to a published procedure (5). The enzyme was taken from the isoelectric focusing column, portions were treated exactly as Solution III, and they were applied to the Amino Acid Analyzer as 0.5-ml aliquots for determination of ninhydrin positive material (8). RESULTS An ampholine solution (pH range 3-10) was separated into three different samples and treated as described above. Table 1 shows the effects TABLE 1 COMPARISON OF DIALYSIS PROCEDURES FOR AMPHOLINE REMOVAL AS OBSERVED ON THE COLUMN FOR SEPARATION OF BASIC AMINO ACIDS
Ampholine residue (mm2)
Percentage remaining
Retention time (rain)"
Untreated
Membrane dialysis
Hollow fiber dialysis
Membrane dialysis
Hollow fiber dialysis
Ratio, Membrane/ hollow fiber
19 29 34 83 88 93 103 Ill 123 131 135 141 176 204 232 258 287 297
1020 3064 43 840 644 624 86 31 144 300 120 45 3278 96 269 85 4521 400
0 688 0 100 104 108 0 0 94 37 20 21 405 0 0 0 227 0
0 292 0 22 22 0 0 0 0 12 0 14 0 0 0 0 46 0
0 23 0 11 16 17 0 0 66 12 17 47 12 0 0 0 5 0
0 10 0 3 3 0 0 0 0 4 0 31 0 0 0 0 1 0
0 2.3 0 3.7 5.3 0 0 3.0 1.5 0 0 0 5.0 0
The retention times for amino acids which may be masked by ampholines are: 29 min, acidic and neutral amino acids; I 11 rain, tryptophan; 149 rain, lysine; 291 rain, arginine.
544
CALLAHAM, POE AND HEITZ
of prior membrane dialysis and hollow fiber dialysis on the ninhydrin positive peaks eluting from the basic amino acid column. The retention times of the peaks separated by the amino acid analyzer are listed in the first column. The ampholines that were retained after the two types of dialysis treatments were compared to the untreated samples in columns 2-4. Percentage retention for each treatment was calculated against the untreated sample, and the results are listed in columns 5 and 6. Finally, the ratio of the percent retention due to membrane dialysis to the percent retention due to hollow fiber dialysis is presented in column 7 as a measure of the effectiveness of hollow fibers over membranes in the removal of ampholine fragments from a solution. In every case, the hollow fibers removed as much or more of the ampholines than did membranes. Table 2 shows the results of the same procedure applied to the effluent of the acidics and neutrals column of the amino acid analyzer. Again, the hollow fibers are shown to be as or more effective in reducing the concentration of fragment ampholines from solutions. Table 3 shows the mean composition of several amino acid analyses of dextransucrase. The standards, e-amino-caproic acid and norleucine, were used to quantitate losses during sample preparation. Since each determination was from a different purification, all residues were normalized with respect to alanine as a standard against purification inequities. The data is presented as percent of total amino acid composition as well as minimum residues per subunit. The minimum molecular weight per subunit based on this data would be approximately 26,000, and the subunit would be composed of 239 residues. TABLE 2 COMPARISON OF DIALYSIS PROCEDURESFOR AMPHOLINE REMOVALAS OBSERVEDON THE COLUMN FOR SEPARATION OF ACIDIC AND NEUTRAL AMINO ACIDS Ampholine residue (ram 2)
Percentage remaining
Retention time (min)a
Untreated
Membrane dialysis
Hollow fiber dialysis
Membrane dialysis
Hollow fiber dialysis
Ratio, Membrane/ hollow fiber
45 62 71 130 156
3705 3749 126 22 21
175 212 0 0 0
70 71 0 0 0
4 5 0 0 0
2 2 0 0 0
2.0 2.5 0 0 0
a The retention times for amino acids which may be masked by ampholines are: 45 min, aspartate; 58 min, serine; 66 rain, glutamate; 134 min, methionine.
545
REMOVAL OF AMPHOLINES TABLE 3 AMINO ACID COMPOSITION OF DEXTRANSUCRASE
Amino acid residue Alanine Arginine Aspartic acid Cysteine/2 Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine
Threonine Tryptophan ~ Tyrosine Valine
Percentage"
Minimum residue/ subunit
8.81 9.67 12.52 0.46 10.41 13.38 2.00 2.69 4.99 10.01 0.42 2.64 2.60 5.81 5.32 0.79 3.26 4.23
2l 23 30 1 25 32 5 6 12 24 1 6 6 14 13 2 8 10
Based on seven determinations for each amino acid except tryptophan. b Based on three determinations by p-toLuene sulfonic acid hydrolysis. "
DISCUSSION Isoelectric focusing has proven to be an effective method of protein purification. One of the major drawbacks to the method concerns the similarity between the ampholine hydrolysis fragments and the amino acids of the protein. For this reason, small molecular weight proteins, such as toxins from various venoms, are unlikely candidates for purification by isoelectric focusing, especially if amino acid analysis is to be required. Larger proteins can be purified by this method and several tehcniques are available for separating the ampholines from the purified protein. The most prominent of these are membrane dialysis, electrophoresis, gel filtration, and salt precipitation. Unfortunately, each of these requires a reasonably large commitment of time and energy since they are purification steps in themselves. It has been shown here that a 1-hr hollow fiber dialysis is more effective at reducing the ampholine concentration than a 24-hr membrane dialysis. With some proteins, not only is the use of hollow fiber dialysis desirable, but it may be essential. Dextransucrase does not easily lend itself to electrophoresis due to its large molecular weight. Gel filtration cannot be used since the enzyme binds tightly to dextran. Salt precipitation is not
546
CALLAHAM, POE AND HEITZ
practical for the low quantities of protein isolated in a typical purification. This study shows that, even though membrane dialysis is capable of removing most of the ampholine fragments, the hollow fiber dialysis is faster and more efficient at removing this unwanted material. Hollow fiber dialysis is therefore recommended for the removal of ampholines from proteins large enough to be retained by the pore-size of the fibers used. The simplicity and efficiency of the technique should be prime considerations in the choice of this procedure. ACKNOWLEDGMENTS This work was supported in part by funds from the Mississippi Agricultural and Forestry Experiment Station, and the Office of Graduate Research, Mississippi State University, Mississippi Agricultural and Forestry Experiment Station Publication No. 3062.
REFERENCES 1. Vesterberg, O., and Svensson, H. (1966)Acta Chem. Scand. 20, 820. 2. Haglund, H. (1971)in Methods of Biochemical Analysis (Glick, D., ed.), Vol. 19, p. 1, Wiley, New York. 3. Vesterberg, O. (1971) In Methods in Enzymology (Jakoby, W. B., ed.) Vol. 22, p. 389, Academic Press, New York. 4. Guggenheim, B., and Newbrun, E. (1969) Heir. Odont. Acta 13, 84. 5. Carlsson, J., Newbrun, E., and Krasse, B. (1%6) Arch. Oral. Biol. 14, 469. 6. Paul, A. B., and Lehmann, I. R. (1%6) J. Biol. Chem. 241, 3441. 7. Liu, T-Y (1972)In Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N., eds.), Vol. 25, p. 44, Academic Press, New York. 8. Spackman, D. H., Stein, W. H., and Moore, S. (1958)Anal. Chem. 30~ 1190.
Removal of Ampholines by Hollow Fiber Dialysis: Application to Dextransucrase M I C H A E L F . C A L L A H A M , W I L L I A M E . POE, AND JAMES R . H E I T Z
Mississippi State University, Mississippi State, Mississippi 39762 Received June 2, 1975; accepted September 2, 1975 Ampholines must he removed from proteins that have been purified using isoelectric focusing chromatography before hydrolysis since ninhydrin positive fragments result. Hollow fiber dialysis was shown to be a fast and efficient method for the removal of ampholines from dextransucrase after isoelectric focusing. The purified protein was then subjected to amino acid analysis.
In enzyme purifications utilizing isoelectric focusing chromatography as one of the methods of purification, it is necessary to remove the ampholines preparatory to determining the amino acid composition of the enzyme. The earliest theories concerning the ease of removal of ampholines by membrane dialysis (1) have been modified by an awareness that the removal by this means may be difficult (2,3). Studies using ampholines alone suggest that the ampholine polymers may be too large to pass the dialysis membrane. Since other techniques of ampholine removal from purified protein, such as gel filtration, electrophoresis, or salt precipitation, may not be applicable in all cases, another alternative method would be desirable. This report is concerned with the use of hollow fiber dialysis as a means of removal of ampholines from dextransucrase, an enzyme that has been purified from bacterial sources using isoelectric focusing chromatography as the terminal step (4,5). This also allows the determination of the amino acid composition of dextransucrase. MATERIALS AND METHODS A 2.5% (v/v) solution o f p H 3 - l0 ampholines (LKB Producter, Sweden) in deionized water was prepared and divided into three parts. Solution I was immediately frozen. Solution II (50 ml) was placed in dialysis tubing, which had been pretreated according to a published procedure (6). Dialysis was carried out for 24 hr against 500 ml of 0.05 M sodium phosphate buffer, pH 6.5, with four buffer changes and constant stirring at 4°C. At the end of this treatment, Solution II was removed and frozen. Solution III was placed in a Beaker Dialyzer (Bio-Rad, Richmond, California) containing cellulose Bio Fiber 50 with a 5000 MW cut off, and the solution was dialyzed. The ampholine solution was placed outside the fibers and 542 Copyright© 1976by AcademicPress, Inc. All rightsof reproductionin any form reserved.
543
REMOVAL OF AMPHOLINES
deionized water, at an approximate flow rate of 200 ml/min, was allowed to pass through the fibers. The dialysis procedure was carried out for 1 hr with constant stirring of the ampholine mixture. This ampholine Solution III was removed and frozen. The three samples were placed in a Virtis Freeze Drying apparatus and lyophilyzed to dryness. Duplicate samples of the three ampholine solutions were hydrolyzed according to a published procedure (7). Dextransucrase was purified according to a published procedure (5). The enzyme was taken from the isoelectric focusing column, portions were treated exactly as Solution III, and they were applied to the Amino Acid Analyzer as 0.5-ml aliquots for determination of ninhydrin positive material (8). RESULTS An ampholine solution (pH range 3-10) was separated into three different samples and treated as described above. Table 1 shows the effects TABLE 1 COMPARISON OF DIALYSIS PROCEDURES FOR AMPHOLINE REMOVAL AS OBSERVED ON THE COLUMN FOR SEPARATION OF BASIC AMINO ACIDS
Ampholine residue (mm2)
Percentage remaining
Retention time (rain)"
Untreated
Membrane dialysis
Hollow fiber dialysis
Membrane dialysis
Hollow fiber dialysis
Ratio, Membrane/ hollow fiber
19 29 34 83 88 93 103 Ill 123 131 135 141 176 204 232 258 287 297
1020 3064 43 840 644 624 86 31 144 300 120 45 3278 96 269 85 4521 400
0 688 0 100 104 108 0 0 94 37 20 21 405 0 0 0 227 0
0 292 0 22 22 0 0 0 0 12 0 14 0 0 0 0 46 0
0 23 0 11 16 17 0 0 66 12 17 47 12 0 0 0 5 0
0 10 0 3 3 0 0 0 0 4 0 31 0 0 0 0 1 0
0 2.3 0 3.7 5.3 0 0 3.0 1.5 0 0 0 5.0 0
The retention times for amino acids which may be masked by ampholines are: 29 min, acidic and neutral amino acids; I 11 rain, tryptophan; 149 rain, lysine; 291 rain, arginine.
544
CALLAHAM, POE AND HEITZ
of prior membrane dialysis and hollow fiber dialysis on the ninhydrin positive peaks eluting from the basic amino acid column. The retention times of the peaks separated by the amino acid analyzer are listed in the first column. The ampholines that were retained after the two types of dialysis treatments were compared to the untreated samples in columns 2-4. Percentage retention for each treatment was calculated against the untreated sample, and the results are listed in columns 5 and 6. Finally, the ratio of the percent retention due to membrane dialysis to the percent retention due to hollow fiber dialysis is presented in column 7 as a measure of the effectiveness of hollow fibers over membranes in the removal of ampholine fragments from a solution. In every case, the hollow fibers removed as much or more of the ampholines than did membranes. Table 2 shows the results of the same procedure applied to the effluent of the acidics and neutrals column of the amino acid analyzer. Again, the hollow fibers are shown to be as or more effective in reducing the concentration of fragment ampholines from solutions. Table 3 shows the mean composition of several amino acid analyses of dextransucrase. The standards, e-amino-caproic acid and norleucine, were used to quantitate losses during sample preparation. Since each determination was from a different purification, all residues were normalized with respect to alanine as a standard against purification inequities. The data is presented as percent of total amino acid composition as well as minimum residues per subunit. The minimum molecular weight per subunit based on this data would be approximately 26,000, and the subunit would be composed of 239 residues. TABLE 2 COMPARISON OF DIALYSIS PROCEDURESFOR AMPHOLINE REMOVALAS OBSERVEDON THE COLUMN FOR SEPARATION OF ACIDIC AND NEUTRAL AMINO ACIDS Ampholine residue (ram 2)
Percentage remaining
Retention time (min)a
Untreated
Membrane dialysis
Hollow fiber dialysis
Membrane dialysis
Hollow fiber dialysis
Ratio, Membrane/ hollow fiber
45 62 71 130 156
3705 3749 126 22 21
175 212 0 0 0
70 71 0 0 0
4 5 0 0 0
2 2 0 0 0
2.0 2.5 0 0 0
a The retention times for amino acids which may be masked by ampholines are: 45 min, aspartate; 58 min, serine; 66 rain, glutamate; 134 min, methionine.
545
REMOVAL OF AMPHOLINES TABLE 3 AMINO ACID COMPOSITION OF DEXTRANSUCRASE
Amino acid residue Alanine Arginine Aspartic acid Cysteine/2 Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine
Threonine Tryptophan ~ Tyrosine Valine
Percentage"
Minimum residue/ subunit
8.81 9.67 12.52 0.46 10.41 13.38 2.00 2.69 4.99 10.01 0.42 2.64 2.60 5.81 5.32 0.79 3.26 4.23
2l 23 30 1 25 32 5 6 12 24 1 6 6 14 13 2 8 10
Based on seven determinations for each amino acid except tryptophan. b Based on three determinations by p-toLuene sulfonic acid hydrolysis. "
DISCUSSION Isoelectric focusing has proven to be an effective method of protein purification. One of the major drawbacks to the method concerns the similarity between the ampholine hydrolysis fragments and the amino acids of the protein. For this reason, small molecular weight proteins, such as toxins from various venoms, are unlikely candidates for purification by isoelectric focusing, especially if amino acid analysis is to be required. Larger proteins can be purified by this method and several tehcniques are available for separating the ampholines from the purified protein. The most prominent of these are membrane dialysis, electrophoresis, gel filtration, and salt precipitation. Unfortunately, each of these requires a reasonably large commitment of time and energy since they are purification steps in themselves. It has been shown here that a 1-hr hollow fiber dialysis is more effective at reducing the ampholine concentration than a 24-hr membrane dialysis. With some proteins, not only is the use of hollow fiber dialysis desirable, but it may be essential. Dextransucrase does not easily lend itself to electrophoresis due to its large molecular weight. Gel filtration cannot be used since the enzyme binds tightly to dextran. Salt precipitation is not
546
CALLAHAM, POE AND HEITZ
practical for the low quantities of protein isolated in a typical purification. This study shows that, even though membrane dialysis is capable of removing most of the ampholine fragments, the hollow fiber dialysis is faster and more efficient at removing this unwanted material. Hollow fiber dialysis is therefore recommended for the removal of ampholines from proteins large enough to be retained by the pore-size of the fibers used. The simplicity and efficiency of the technique should be prime considerations in the choice of this procedure. ACKNOWLEDGMENTS This work was supported in part by funds from the Mississippi Agricultural and Forestry Experiment Station, and the Office of Graduate Research, Mississippi State University, Mississippi Agricultural and Forestry Experiment Station Publication No. 3062.
REFERENCES 1. Vesterberg, O., and Svensson, H. (1966)Acta Chem. Scand. 20, 820. 2. Haglund, H. (1971)in Methods of Biochemical Analysis (Glick, D., ed.), Vol. 19, p. 1, Wiley, New York. 3. Vesterberg, O. (1971) In Methods in Enzymology (Jakoby, W. B., ed.) Vol. 22, p. 389, Academic Press, New York. 4. Guggenheim, B., and Newbrun, E. (1969) Heir. Odont. Acta 13, 84. 5. Carlsson, J., Newbrun, E., and Krasse, B. (1%6) Arch. Oral. Biol. 14, 469. 6. Paul, A. B., and Lehmann, I. R. (1%6) J. Biol. Chem. 241, 3441. 7. Liu, T-Y (1972)In Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N., eds.), Vol. 25, p. 44, Academic Press, New York. 8. Spackman, D. H., Stein, W. H., and Moore, S. (1958)Anal. Chem. 30~ 1190.
