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[14] Purification of avidin and its derivatives on 2-iminobiotin-6-aminohexyl-sepharose 4B
Cl41
AFFINITY
PURIFICATION
83
OF AVIDIN
1141 Purification of Avidin and Its Derivatives on 2-Iminobiotin-6-aminohexyl-Sepharose 4B By GEORGE
A.
ORR,
GAYLE
C.
HENEY,
and RON
ZEHEB
Principle The avidin-biotin complex possesses an extremely low dissociation constant (&) of approximately lo-Is M.' The tightness of this interaction has formed the basis for the use of the complex in several techniques in membrane and molecular biology. 2*3In many of these studies, it would be desirable if an affinity isolation system were available for the isolation of biologically active avidin derivatives that were uncontaminated by damaged avidin or unconjugated reporter molecules. Cuatrecasas and Wilchek have reported the isolation of avidin on a biocytin (biotin-e-N-lysyl)Sepharose 4B column.4 The conditions required for the elution of the specifically bound protein were a combination of low pH (I S) and 6 M guanidine-HCI; either alone did not effect elution. Although avidin is not inactivated by these harsh elution conditions, it is uncertain whether certain avidin derivatives, e.g., horseradish peroxidase or alkaline phosphatase conjugated avidin, would retain their desired biological activity. We have found,s in agreement with the observations of Green,’ that the free base form of 2-iminobiotin forms a stable complex with avidin, but that the salt form interacts poorly with its binding protein (Fig. 1). Our studies indicate that the decrease in affinity observed at neutral and acidic pH values is due to the combined protonation of the cyclic guanidino group of 2-iminobiotin and the ionization of some residue on avidin. We have used this pH-dependent alteration in binding to develop an efficient affinity isolation procedure for avidin and its derivatives.6 Synthesis of 2-Iminobiotin Hydrobromide The two-step synthesis involves the alkaline hydrolysis of biotin with barium hydroxide at 140”yielding 5-(3,4-diaminothiophan-2-yl)pentanoic ’ N. M. Green, Adu. Protein Chem. 29, 85 (1975). 2 E. A. Bayer and M. Wilchek Trends Biochem. Sci. 3, N257 (1978). 3 E. A. Bayer and M. Wilchek, this series, Vol. 62, p. 308. 4 P. Cuatrecasas and M. Wilchek, Biochem. Biophys. Res. Commun. 33, 235 (1%8). 5 G. A. Orr, J. Biol. Chem. 256, 761 (1980). 6 G. Heney and G. A. Orr, Anal. Biochem. 114,92 (1981).
METHODS
IN ENZYMOLOGY,
VOL.
122
Copyright 0 1986 by Academic Press, Lnc. All rights of reproduction in any form reserved.
84
BIOTIN
NHz
AND
r141
DERIVATIVES
pK,ll-12
0’ ‘I ,,*&kNH -HO
NH II H N/‘\NH
+HO (CH&COOH
K,j < 10-3M
P3-f2 ),COOH Kd=3.5X1o-“M
FIG. 1. Structure of 2-iminobiotin showing ionization of the cyclic guanidino group.
acid.7 2-Iminobiotin is subsequently prepared from this diaminocarboxylic acid derivative of biotin by reaction with cyanogen bromide.8 5(3,4-Diaminothiophan-2-yl)pentanoic acid. Biotin (1 g) and barium hydroxide (3 g) are mixed together and placed into a Pyrex hydrolysis tube (I5 x 250 mm). Water (7 ml) is added and the tube sealed under mild vacuum (house vacumm line, approx 30 mm Hg). After heating at 140”for 21 hr, the tube contents are removed, CO2 is bubbled into the suspension, and the insoluble precipitate (BaC03) is removed by filtration. The filtrate is acidified to pH 4 using 2 N H2S04, filtered, and concentrated in uacuo. Addition of methanol induces crystallization of the diaminocarboxylic acid sulfate and this process is allowed to continue overnight at 40”(56% yield). Thin-layer chromatography on silica gel (CHCls : Hz0 : concentrated NH40H, 75 : 25 : 5) reveals the presence of a single I*- and ninhydrin-positive spot of Rf 0.6. 2-Zminobiotin. The diaminocarboxylic acid sulfate (2.5 g) is treated with barium carbonate (5 g) and dissolved in 30 ml of hot H20, and the solution immediately filtered through a prewarmed fritted glass funnel. The precipitate is washed with an additional 10 ml of hot H20. Cyanogen bromide (1.9 g) is added to the filtrate, and crystals of 2-iminobiotin (free base form) start to form within 5 min. The reaction mixture is left at 4”for 12 hr. The yield of 2-iminobiotin is 1.8 g (m.p. > 260” decomp.). To convert the free base into the hydrobromide salt, 2-iminobiotin is suspended in H20 (40 ml) and heated to approximately 45”, and 1% HBr is added dropwise until all of the 2-iminobiotin dissolves. The solution is concentrated in uacuo and the residue recrystallized from hot 2-propanolmethanol (6: 4, v/v). 2-Iminobiotin hydrobromide has a melting point of 222-223”. Thin-layer chromatography on silica gel eluting with CHC& : Hz0 : concentrated NH40H(70 : 25 : 5, v/v) reveals the presence of a single 12-positive spot with an Rf of 0.38. ’ K. Hofmann, D. B. Melville, and V. Du Vigneaud, J. Biol. Chem. 141, 207 (1941). * K. Hofmann and A. E. Axelrod, J. Biol. Chem. 187, 29 (1950).
I141
AFFINITY
PURIFICATION
OF AVIDIN
85
Purification of Avidin 2-Iminobiotin is coupled to 6-aminohexyl-Sepharose 4B using a watersoluble carbodiimide. Although 2-iminobiotin contains a potentially reactive guanidino group (pK, ll-12), this does not complicate the coupling, since the reaction is carried out at pH 4.8, where the group is fully protonated. Purified avidin is bound by the affinity matrix at pH I I and is eluted as a sharp peak when the pH is lowered to 4. Biotin-treated avidin is not retained by the affinity column, indicating that binding is specific to the 2-iminobiotin moiety. For efficient binding to immobilized 2-iminobiotin, the pH must be greater than 9.0. We have used this affinity matrix for the isolation of avidin from homogenized egg whites in a single step. However, for the purification of large amounts of avidin, we have found it more convenient to carry out a preliminary ammonium sulfate fractionation. 2-Iminobiotin-6-aminohexyl-Sepharose 4B. 2-Iminobiotin (400 mg, 1.23 mmol) is added to 100 ml of 6-aminohexyl-Sepharose 4B (40 ml of packed resin in H20), prepared by the method of Porath,9 and the pH adjusted to 4.8 with HBr (I%, v/v). I-Cyclohexyl-3-(2-morpholinoethyl)carbodiimidemetho-p-toluene sulfonate (4.24 g, 10 mmol) is added portionwise over a period of 10 min. The pH is kept at 4.8 throughout the reaction by the addition of 1% HBr and is constant after 3-5 hr. The resin is washed with 1 M NaCl (2 liters) and Hz0 (2 liters) and packed into a column. The binding capacity of the affinity matrix, using these coupling conditions is 0.75 mg purified avidin/ml of swollen gel. Affinity Isolation of A&in. Homogenized egg whites from 24 fresh jumbo eggs are diluted with HZ0 (2 : 1, v/v) and the solution is brought to 70% saturation with ammonium sulfate (enzyme grade, Schwartz/Mann) at 4”. After stirring for 2 hr, the mixture is centrifuged (8000g, 20 min), the supernatant brought to 100% saturation, and the mixture left stirring at 4 overnight. After centrifugation (8000 g, 30 min), the pellet is dissolved in Hz0 (40 ml) and dialyzed against Hz0 (3 x 2 liters). The pH of the dialysate is adjusted to 11 with 1 N NaOH and NaCl (1 M) added. The crude avidin solution is applied to 2-iminobiotin-6-aminohexyl-Sepharose 4B (40 ml) which had previously been equilibrated with 50 mM sodium carbonate (pH 11) containing 1 M NaCl. The column is washed with equilibrating buffer (20 ml/hr) until the absorbance at 282 nm returns to baseline. Avidin is eluted from the column with 50 mM ammonium acetate (pH 4) containing 0.5 M NaCl. Protein content is measured by absorbance at 282 nm and avidin content by its ability to bind either 4-hy9 J. Porftth, this series, Vol. 18 Part B,
p. 13.
86
BIOTIN
AND
r141
DERIVATIVES
3
2
2.8
2
2.4 _
2.0 %
% 2 1 E
1.6 2 .
c5 1.
1.2 2
8 6O
0.8 g 5 0.4 6
0
FRACTION NC,
FIG. 2. AfIinity purification of avidin on 2-iminobiotin-6-aminohexyl-Sepharose 4B. Crude avidin was loaded onto the column at pH 11 and specifically eluted by application of pH 4 buffer.
droxyazobenzene-2’carboxylic acid or [14C]biotin.10 The appropriate fractions are pooled, dialyzed against H20, and lyophilized. As can be seen from Fig. 2, avidin is eluted as a sharp peak after application of the low pH buffer. Greater than 90% of the crude avidin applied to the column is recovered in the specifically eluted fractions, and the yield of avidin from 24 eggs is in the range of 15-20 mg. We have used the same affinity column for several such avidin preparations with no apparent loss in activity. The avidin obtained by this procedure is pure as judged by its ability to bind 14.4 pg of [14C]biotin/mg of protein. Literature values for pure avidin range from 13.8 to 15.1.*s4SDS-polyacrylamide gel electrophoresis of the specifically eluted fractions reveals a single polypeptide with an apparent molecular weight slightly larger than the hemoglobin monomer (16,000). Native avidin (68,000) is composed of four identical subunits.’ As a further indication of purity, if the specifically eluted protein is treated with biotin and rechromatographed on the affinity column, no protein is retained and eluted at pH 4. Conclusions The pH-dependent interaction between 2-iminobiotin and avidin has enabled us to develop an affinity isolation procedure for avidin which lo N. M. Green, this series, Vol. 18 Part A, p. 418,
1151
AVIDIN-IMINOBIOTIN-BASED
PROTEIN
PURIFICATION
87
overcomes the harsh elution condition associated with immobilized biotin columns. We have also used this system for the purification of both I*% and rhodamine-labeled avidin. This method should also prove suitable for the purification of enzyme-conjugated avidin derivatives, e.g., horseradish peroxidase and alkaline phosphates conjugates. Acknowledgments This research was supported in part by Grants GM 27851, GM 34029, and Cancer Core Grant P30-CA13330. G.A.O. is a recipient of a Research Career Development Award (HD 00577). R.Z. was supported in part by NIH Training Grant 5T32-GM 07260.
[151 Use of Avidin-Iminobiotin
Complexes Plasma Membrane Proteins
for Purifying
By RON ZEHEB and GEORGE A. ORR Principle
An analytical and preparative approach which provides information concerning the organization and function of cell surface components without the prior isolation of plasma membranes has been developed.‘,* The basis of the technique is the covalent attachment of compounds containing 2-iminobiotin, the cyclic guanidino analog of biotin, onto cell surface proteins. The “tagged” species are then isolated by virtue of the unique interaction between the covalently attached ligand and its binding protein, avidin. The pH-dependent interaction of 2-iminobiotin with avidin makes recovery possible. At high pH, the free base form of 2-iminobiotin retains the high-affinity specific binding to avidin characteristic of biotin (& = 10-l’ M) whereas at acidic pH values, the salt form of the analog interacts poorly with avidin (Kd > 10e3M). By chemically modifying the valeric acid side chain of 24minobiotin (Fig. I), it is possible to create a series of molecules capable of covalently “tagging” membrane proteins and glycoproteins with 2-iminobiotin. The specific site of covalent attachment can be varied depending on the congener of 2-iminobiotin used and the choice of prior chemical or enzymatic treatment of the target membranes. Additionally, the method permits labeling under native conditions in which cell membrane integrity is maintained. ’ G. A. Orr, J. Biol. Chem. 256,761 (1981). 2 R. Zeheb, V. Chang, and G. A. Orr, Anal. Biochem. 129, 156 (1983).
METHODS
IN ENZYMOLOGY,
VOL.
122
Copyright Q 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
AFFINITY
PURIFICATION
83
OF AVIDIN
1141 Purification of Avidin and Its Derivatives on 2-Iminobiotin-6-aminohexyl-Sepharose 4B By GEORGE
A.
ORR,
GAYLE
C.
HENEY,
and RON
ZEHEB
Principle The avidin-biotin complex possesses an extremely low dissociation constant (&) of approximately lo-Is M.' The tightness of this interaction has formed the basis for the use of the complex in several techniques in membrane and molecular biology. 2*3In many of these studies, it would be desirable if an affinity isolation system were available for the isolation of biologically active avidin derivatives that were uncontaminated by damaged avidin or unconjugated reporter molecules. Cuatrecasas and Wilchek have reported the isolation of avidin on a biocytin (biotin-e-N-lysyl)Sepharose 4B column.4 The conditions required for the elution of the specifically bound protein were a combination of low pH (I S) and 6 M guanidine-HCI; either alone did not effect elution. Although avidin is not inactivated by these harsh elution conditions, it is uncertain whether certain avidin derivatives, e.g., horseradish peroxidase or alkaline phosphatase conjugated avidin, would retain their desired biological activity. We have found,s in agreement with the observations of Green,’ that the free base form of 2-iminobiotin forms a stable complex with avidin, but that the salt form interacts poorly with its binding protein (Fig. 1). Our studies indicate that the decrease in affinity observed at neutral and acidic pH values is due to the combined protonation of the cyclic guanidino group of 2-iminobiotin and the ionization of some residue on avidin. We have used this pH-dependent alteration in binding to develop an efficient affinity isolation procedure for avidin and its derivatives.6 Synthesis of 2-Iminobiotin Hydrobromide The two-step synthesis involves the alkaline hydrolysis of biotin with barium hydroxide at 140”yielding 5-(3,4-diaminothiophan-2-yl)pentanoic ’ N. M. Green, Adu. Protein Chem. 29, 85 (1975). 2 E. A. Bayer and M. Wilchek Trends Biochem. Sci. 3, N257 (1978). 3 E. A. Bayer and M. Wilchek, this series, Vol. 62, p. 308. 4 P. Cuatrecasas and M. Wilchek, Biochem. Biophys. Res. Commun. 33, 235 (1%8). 5 G. A. Orr, J. Biol. Chem. 256, 761 (1980). 6 G. Heney and G. A. Orr, Anal. Biochem. 114,92 (1981).
METHODS
IN ENZYMOLOGY,
VOL.
122
Copyright 0 1986 by Academic Press, Lnc. All rights of reproduction in any form reserved.
84
BIOTIN
NHz
AND
r141
DERIVATIVES
pK,ll-12
0’ ‘I ,,*&kNH -HO
NH II H N/‘\NH
+HO (CH&COOH
K,j < 10-3M
P3-f2 ),COOH Kd=3.5X1o-“M
FIG. 1. Structure of 2-iminobiotin showing ionization of the cyclic guanidino group.
acid.7 2-Iminobiotin is subsequently prepared from this diaminocarboxylic acid derivative of biotin by reaction with cyanogen bromide.8 5(3,4-Diaminothiophan-2-yl)pentanoic acid. Biotin (1 g) and barium hydroxide (3 g) are mixed together and placed into a Pyrex hydrolysis tube (I5 x 250 mm). Water (7 ml) is added and the tube sealed under mild vacuum (house vacumm line, approx 30 mm Hg). After heating at 140”for 21 hr, the tube contents are removed, CO2 is bubbled into the suspension, and the insoluble precipitate (BaC03) is removed by filtration. The filtrate is acidified to pH 4 using 2 N H2S04, filtered, and concentrated in uacuo. Addition of methanol induces crystallization of the diaminocarboxylic acid sulfate and this process is allowed to continue overnight at 40”(56% yield). Thin-layer chromatography on silica gel (CHCls : Hz0 : concentrated NH40H, 75 : 25 : 5) reveals the presence of a single I*- and ninhydrin-positive spot of Rf 0.6. 2-Zminobiotin. The diaminocarboxylic acid sulfate (2.5 g) is treated with barium carbonate (5 g) and dissolved in 30 ml of hot H20, and the solution immediately filtered through a prewarmed fritted glass funnel. The precipitate is washed with an additional 10 ml of hot H20. Cyanogen bromide (1.9 g) is added to the filtrate, and crystals of 2-iminobiotin (free base form) start to form within 5 min. The reaction mixture is left at 4”for 12 hr. The yield of 2-iminobiotin is 1.8 g (m.p. > 260” decomp.). To convert the free base into the hydrobromide salt, 2-iminobiotin is suspended in H20 (40 ml) and heated to approximately 45”, and 1% HBr is added dropwise until all of the 2-iminobiotin dissolves. The solution is concentrated in uacuo and the residue recrystallized from hot 2-propanolmethanol (6: 4, v/v). 2-Iminobiotin hydrobromide has a melting point of 222-223”. Thin-layer chromatography on silica gel eluting with CHC& : Hz0 : concentrated NH40H(70 : 25 : 5, v/v) reveals the presence of a single 12-positive spot with an Rf of 0.38. ’ K. Hofmann, D. B. Melville, and V. Du Vigneaud, J. Biol. Chem. 141, 207 (1941). * K. Hofmann and A. E. Axelrod, J. Biol. Chem. 187, 29 (1950).
I141
AFFINITY
PURIFICATION
OF AVIDIN
85
Purification of Avidin 2-Iminobiotin is coupled to 6-aminohexyl-Sepharose 4B using a watersoluble carbodiimide. Although 2-iminobiotin contains a potentially reactive guanidino group (pK, ll-12), this does not complicate the coupling, since the reaction is carried out at pH 4.8, where the group is fully protonated. Purified avidin is bound by the affinity matrix at pH I I and is eluted as a sharp peak when the pH is lowered to 4. Biotin-treated avidin is not retained by the affinity column, indicating that binding is specific to the 2-iminobiotin moiety. For efficient binding to immobilized 2-iminobiotin, the pH must be greater than 9.0. We have used this affinity matrix for the isolation of avidin from homogenized egg whites in a single step. However, for the purification of large amounts of avidin, we have found it more convenient to carry out a preliminary ammonium sulfate fractionation. 2-Iminobiotin-6-aminohexyl-Sepharose 4B. 2-Iminobiotin (400 mg, 1.23 mmol) is added to 100 ml of 6-aminohexyl-Sepharose 4B (40 ml of packed resin in H20), prepared by the method of Porath,9 and the pH adjusted to 4.8 with HBr (I%, v/v). I-Cyclohexyl-3-(2-morpholinoethyl)carbodiimidemetho-p-toluene sulfonate (4.24 g, 10 mmol) is added portionwise over a period of 10 min. The pH is kept at 4.8 throughout the reaction by the addition of 1% HBr and is constant after 3-5 hr. The resin is washed with 1 M NaCl (2 liters) and Hz0 (2 liters) and packed into a column. The binding capacity of the affinity matrix, using these coupling conditions is 0.75 mg purified avidin/ml of swollen gel. Affinity Isolation of A&in. Homogenized egg whites from 24 fresh jumbo eggs are diluted with HZ0 (2 : 1, v/v) and the solution is brought to 70% saturation with ammonium sulfate (enzyme grade, Schwartz/Mann) at 4”. After stirring for 2 hr, the mixture is centrifuged (8000g, 20 min), the supernatant brought to 100% saturation, and the mixture left stirring at 4 overnight. After centrifugation (8000 g, 30 min), the pellet is dissolved in Hz0 (40 ml) and dialyzed against Hz0 (3 x 2 liters). The pH of the dialysate is adjusted to 11 with 1 N NaOH and NaCl (1 M) added. The crude avidin solution is applied to 2-iminobiotin-6-aminohexyl-Sepharose 4B (40 ml) which had previously been equilibrated with 50 mM sodium carbonate (pH 11) containing 1 M NaCl. The column is washed with equilibrating buffer (20 ml/hr) until the absorbance at 282 nm returns to baseline. Avidin is eluted from the column with 50 mM ammonium acetate (pH 4) containing 0.5 M NaCl. Protein content is measured by absorbance at 282 nm and avidin content by its ability to bind either 4-hy9 J. Porftth, this series, Vol. 18 Part B,
p. 13.
86
BIOTIN
AND
r141
DERIVATIVES
3
2
2.8
2
2.4 _
2.0 %
% 2 1 E
1.6 2 .
c5 1.
1.2 2
8 6O
0.8 g 5 0.4 6
0
FRACTION NC,
FIG. 2. AfIinity purification of avidin on 2-iminobiotin-6-aminohexyl-Sepharose 4B. Crude avidin was loaded onto the column at pH 11 and specifically eluted by application of pH 4 buffer.
droxyazobenzene-2’carboxylic acid or [14C]biotin.10 The appropriate fractions are pooled, dialyzed against H20, and lyophilized. As can be seen from Fig. 2, avidin is eluted as a sharp peak after application of the low pH buffer. Greater than 90% of the crude avidin applied to the column is recovered in the specifically eluted fractions, and the yield of avidin from 24 eggs is in the range of 15-20 mg. We have used the same affinity column for several such avidin preparations with no apparent loss in activity. The avidin obtained by this procedure is pure as judged by its ability to bind 14.4 pg of [14C]biotin/mg of protein. Literature values for pure avidin range from 13.8 to 15.1.*s4SDS-polyacrylamide gel electrophoresis of the specifically eluted fractions reveals a single polypeptide with an apparent molecular weight slightly larger than the hemoglobin monomer (16,000). Native avidin (68,000) is composed of four identical subunits.’ As a further indication of purity, if the specifically eluted protein is treated with biotin and rechromatographed on the affinity column, no protein is retained and eluted at pH 4. Conclusions The pH-dependent interaction between 2-iminobiotin and avidin has enabled us to develop an affinity isolation procedure for avidin which lo N. M. Green, this series, Vol. 18 Part A, p. 418,
1151
AVIDIN-IMINOBIOTIN-BASED
PROTEIN
PURIFICATION
87
overcomes the harsh elution condition associated with immobilized biotin columns. We have also used this system for the purification of both I*% and rhodamine-labeled avidin. This method should also prove suitable for the purification of enzyme-conjugated avidin derivatives, e.g., horseradish peroxidase and alkaline phosphates conjugates. Acknowledgments This research was supported in part by Grants GM 27851, GM 34029, and Cancer Core Grant P30-CA13330. G.A.O. is a recipient of a Research Career Development Award (HD 00577). R.Z. was supported in part by NIH Training Grant 5T32-GM 07260.
[151 Use of Avidin-Iminobiotin
Complexes Plasma Membrane Proteins
for Purifying
By RON ZEHEB and GEORGE A. ORR Principle
An analytical and preparative approach which provides information concerning the organization and function of cell surface components without the prior isolation of plasma membranes has been developed.‘,* The basis of the technique is the covalent attachment of compounds containing 2-iminobiotin, the cyclic guanidino analog of biotin, onto cell surface proteins. The “tagged” species are then isolated by virtue of the unique interaction between the covalently attached ligand and its binding protein, avidin. The pH-dependent interaction of 2-iminobiotin with avidin makes recovery possible. At high pH, the free base form of 2-iminobiotin retains the high-affinity specific binding to avidin characteristic of biotin (& = 10-l’ M) whereas at acidic pH values, the salt form of the analog interacts poorly with avidin (Kd > 10e3M). By chemically modifying the valeric acid side chain of 24minobiotin (Fig. I), it is possible to create a series of molecules capable of covalently “tagging” membrane proteins and glycoproteins with 2-iminobiotin. The specific site of covalent attachment can be varied depending on the congener of 2-iminobiotin used and the choice of prior chemical or enzymatic treatment of the target membranes. Additionally, the method permits labeling under native conditions in which cell membrane integrity is maintained. ’ G. A. Orr, J. Biol. Chem. 256,761 (1981). 2 R. Zeheb, V. Chang, and G. A. Orr, Anal. Biochem. 129, 156 (1983).
METHODS
IN ENZYMOLOGY,
VOL.
122
Copyright Q 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.