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[56] Egg yolk biotin-binding protein: Assay and purification
316
BIOTIN AND DERIVATIVES
[56]
[56] E g g Y o l k B i o t i n - B i n d i n g P r o t e i n : Assay and Purification B y HARRY W . MESLAR a n d HAROLD B . W H I T E , III
Assay Method
Principle. The assay is based on the temperature-accelerated exchange of bound biotin for free 1-14C]biotin.1 The procedure is, therefore, useful for biotin-binding proteins with no free binding sites, s Reagents Potassium phosphate, 50 mM, pH 7.2 [14C]Biotin, 15/zg/ml, 20/zCi/mg in pH 7.2 buffer 3 Bentonite, 10 mg/ml suspension in pH 7.2 buffer Bray's solution, liquid scintillation fluid 4 Definition of Unit. One unit is defined as the amount of protein that will bind 1/xg of d-biotin. Procedure. A sample, containing 2.6 × 104 to 2.6 × 10-n units (2-20 /zg of egg yolk biotin-binding protein or avidin) is pipetted into a test tube. A 50-tzl portion of [J4C]biotin is added, and the mixture is incubated for 15 min at the appropriate temperature (65 ° for yolk biotin-binding protein, 85 ° for avidin). The sample is cooled on ice, and 300/zl of the bentonite suspension is added to adsorb the protein. The slurry is poured into a vacuum-sampling manifold equipped with nitrocellulose filters and washed 3 times with 1 ml of cold 50 mM potassium phosphate buffer, pH 7.2. After the last wash has dried, the filters are dissolved in scintillation fluid and the bound radioactivity is measured in a liquid scintillation counter. Blank values are obtained by running a series of assays at different protein concentrations and extrapolating to zero protein. The filtering of materials of high lipid content (e.g., egg yolk) is greatly aided by replacing the first buffer wash with 1 ml of water-saturated 1-butanol. The butanol dissolves the lipids without damaging the protein. I H. B. White, B. A. Dennison, M. A. DellaFera, C. J. Whitney, J. C. McGuire, H. W. Meslar, and P. H. Sammelwitz, Biochem. J. 157, 396 (1976). 2 For assay of avidin with available binding sites, see N. M. Green, this series, Vol. 18A, p. 414; R. Wei, this series, Vol. 18A, p. 424; S. G. Korenman and B. W. O'MaUey, this series, Voi. 18A, p. 427. a In our experience 30-50% of the radioactivity in commercially prepared [~4C~oiotin will not bind to avidin. The specific activity of samples diluted with unlabeled biotin must be adjusted to compensate for these impurities. 4 G. A. Bray, Anal. Biochem. 1, 279 (1960).
METHODS IN ENZYMOLOGY. VOL. 62
Copyright© 1979by AcademicPress.Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181962-0
[56]
EGG YOLK BIOTIN-BINDING PROTEIN
317
Purification Procedure 5
General. All buffers used are potassium phosphate, pH 7.2, at the concentration given. The procedure outlined is for 5 dozen eggs. Lipid Removal. e The eggs are cracked and the white and yolks are separated. The yolks are broken and 1.5 times the yolk volume in distilled water is added. The diluted yolk is well stirred. This solution is centrifuged at 13,200 g for 30 min. The supernatant is collected, and solid NaCl is added to make the solution 1 M in salt. After the salt has completely dissolved, the solution is poured into centrifuge bottles, two-thirds the solution volume in 1-butanol is added, and the mixture is shaken vigorously.r Centrifugation of the resulting emulsion at 13,200 g for 15 min causes a lipid-free aqueous phase and a lipid-rich organic phase to form, with a solid layer of precipitated protein at the interface. The two liquids are poured into a separatory funnel and the lower aqueous phase is removed. The aqueous phase is dialyzed overnight against running water at 4° to remove NaC1 and butanol. Phosphocellulose Ion-Exchange Chromatography. To the dialyzed lipid-free yolk solution is added 150 ml of settled phosphocellulose, equilibrated with 50 mM potassium phosphate, pH 7.2, and the suspension is stirred. The phosphocellulose is allowed to settle, and the supernatant is decanted. The phosphocellulose is poured into a column (2.5 × 50 cm) attached to a peristaltic pump and a UV column monitor. The column is washed with 50 mM potassium phosphate until the ODzs0 of the eluate has regained baseline. The flow is discontinued, and the 50 mM buffer is replaced by 0.5 M potassium phosphate. The pump is started, and the sharp peak of protein eluted is collected and pooled. Affinity Chromatography. The phosphocellulose eluate is placed in a pressure concentrator equipped with an ultrafiltering membrane (0.45 /zm) along with l0 ml of biotinyl-Sepharose, s The total volume must not exceed 150 ml in a 400-ml concentrator. The concentrator is lowered into a 55 ° water bath equipped for magnetic stirring° and pressurized with nitrogen, taking care not to increase the volume in the concentrator. The af5 H. W. Meslar, S. A. Camper, and H. B. White, III, J. Biol. Chem. 253, 6979 (1978). 6 H. W. Meslar and H. B. White, III, Anal. Biochem. 91, 75 (1978). 7 R. K. Morton, this series, Vol. l, p. 40. a Biotinyl-Sepharose is prepared by water-soluble carbodiimide coupling of biotin to hexamethylene-diamine substituted Sepharose 4BCL. The coupling must take place at pH 5.7-6.0. Below pH 5.7 the biotin becomes insoluble, and pH 6.0 is the upper limit for successful carbodiimide coupling. See I. Parikh, S. March, and P. Cuatrecasas, this series, Vol. 34B, p. 77. a An aluminum pot on top of a heater-stirrer has been used quite successfully in our laboratory for this purpose.
318
BIOTIN AND DERIVATIVES
[56]
SUMMARY OF PURIFICATION PROCEDURE
Fraction
Total units
Specific activity (units/mg)a
Diluted yolk Phosphocellulose eluate Gel filtration eluate b
378 58.6 5.25
6.82 × 10-3 1.42 12-13
a Protein determinations were made using the Bio-Rad dye binding assay kit, Bio-Rad Laboratories, Richmond, California. b Activity of the affinity gel eluate is not measured because of the excess unlabeled biotin present at that stage.
finity gel-protein slurry is washed with 2.0-2.3 liters of 50 mM potassium phosphate and depressurized. The gel is poured into a water-jacketed column (1.0 × 15 cm) and washed with 0.3 M potassium phosphate until the OD~0 of the eluate reaches baseline. The water jacket is filled with water at 65 °, and the column is filled with 10 ml of 40 mM biotin, dissolved in 0.2 M potassium phosphate. The system is allowed to heat and then is slowly eluted (0.6 ml/min). The entire biotin-containing peak is pooled. Gel Filtration. The eluted material is concentrated to a volume of 2 ml, made dense with solid sucrose, and applied to a column of Ultrogel AcA 44, l° equilibrated with 0.2 M potassium phosphate, and eluted. Fractions of constant specific activity are pooled and lyophilized. Summary of the Purification. The procedure is summarized in the table. This procedure has been successfully repeated 10 times. The protein shows a single band on gel electrophoresis, shows a single N-terminal, and gives a single precipitin band with antiserum raised against the mixture of proteins eluted from phosphocellulose. Properties
Stability. The protein is unstable in dilute solution at low ionic strength (50 mM potassium phosphate). At higher ionic strength, it is stable for months when frozen. The protein denatures at temperatures above 70° . Molecular Structure. Egg yolk biotin-binding protein appears to be a tetramer of identical subunits. The molecular weight of the tetramer is about 74,300. pH Optimum. Biotin is tightly bound from pH 5 to above pH 10. Below pH 5.0, the biotin-binding activity decreases and at pH 2.3 the protein is completely inactive. ~o LKB Instruments, RockviUe, Maryland.
[571
BIOTIN-BINDING ANTIBODIES
3 19
Temperature-Induced Biotin Release. Yolk biotin-binding protein has a maximum rate of biotin release at 70 °, above which temperature the protein begins to denature. Comparison with Egg White Avidin. 5 The properties of yolk biotinbinding protein are very different than those reported for avidin. 11 The pI is about pH 4.6, as opposed to about pH 10 for avidin. The yolk protein, although heavier (74,300 as opposed to 68,000), contains fewer residues of lysine, threonine, isoleucine, and aspartic acid. Biotin release is more than 1000-fold faster from the egg yolk protein than from the egg white protein. Neither protein cross-reacts with antibodies directed against the other. Cyanogen bromide cleavage generates peptides containing Nterminal leucine from egg yolk biotin-binding protein and N-terminal threonine and tryptophan from avidin. 11 N. M. Green, Adv. Protein Chem. 29, 85 (1975).
[57] A n t i b o d i e s T h a t B i n d B i o t i n a n d Inhibit Biotin-Containing Enzymes a By M E L V I N BERGER
Antibodies with affinity for biotin are produced following immunization of rabbits with antigens consisting of biotin linked covalently as a hapten to a carrier protein such as bovine serum albumin or bovine y-globulin, z In recent years considerable technology has been developed for the use of specific antibodies as biochemical reagents, a'4 Antibodies that bind biotin thus could be very useful in the purification and analysis of biotin-containing enzymes. In particular, the Fab fragments of these antibodies could be used as specific, monovalent, biotin-binding proteins, offering an advantage over the tetravalent avidin molecule in some cases. The avidity of these antibodies for enzyme-bound biotin allows enzyme inhibition to be used as a simple assay for the production of biotin-binding antibody. Enzyme inhibition can also serve as a convenient measure of unsaturated binding sites in studies of the interaction of biotin or its derivatives with the antibodies. I This work was supported by National Institutes of Health Grants Nos. 5T01-GM-00035 and AM-12245 and was performed in the laboratory of Dr. Harland G. Wood in the Department of Biochemistry, Case Western Reserve University. 2 M. Berger, Biochemistry 14, 2338 (1975). 3 j. B. Robbins and R. Schneerson, this series, Vol. 34, p. 703. 4 D. M. Livingston this series, Vol. 34, p. 723.
METHODS IN ENZYMOLOGY, VOL. 62
Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181962-0
BIOTIN AND DERIVATIVES
[56]
[56] E g g Y o l k B i o t i n - B i n d i n g P r o t e i n : Assay and Purification B y HARRY W . MESLAR a n d HAROLD B . W H I T E , III
Assay Method
Principle. The assay is based on the temperature-accelerated exchange of bound biotin for free 1-14C]biotin.1 The procedure is, therefore, useful for biotin-binding proteins with no free binding sites, s Reagents Potassium phosphate, 50 mM, pH 7.2 [14C]Biotin, 15/zg/ml, 20/zCi/mg in pH 7.2 buffer 3 Bentonite, 10 mg/ml suspension in pH 7.2 buffer Bray's solution, liquid scintillation fluid 4 Definition of Unit. One unit is defined as the amount of protein that will bind 1/xg of d-biotin. Procedure. A sample, containing 2.6 × 104 to 2.6 × 10-n units (2-20 /zg of egg yolk biotin-binding protein or avidin) is pipetted into a test tube. A 50-tzl portion of [J4C]biotin is added, and the mixture is incubated for 15 min at the appropriate temperature (65 ° for yolk biotin-binding protein, 85 ° for avidin). The sample is cooled on ice, and 300/zl of the bentonite suspension is added to adsorb the protein. The slurry is poured into a vacuum-sampling manifold equipped with nitrocellulose filters and washed 3 times with 1 ml of cold 50 mM potassium phosphate buffer, pH 7.2. After the last wash has dried, the filters are dissolved in scintillation fluid and the bound radioactivity is measured in a liquid scintillation counter. Blank values are obtained by running a series of assays at different protein concentrations and extrapolating to zero protein. The filtering of materials of high lipid content (e.g., egg yolk) is greatly aided by replacing the first buffer wash with 1 ml of water-saturated 1-butanol. The butanol dissolves the lipids without damaging the protein. I H. B. White, B. A. Dennison, M. A. DellaFera, C. J. Whitney, J. C. McGuire, H. W. Meslar, and P. H. Sammelwitz, Biochem. J. 157, 396 (1976). 2 For assay of avidin with available binding sites, see N. M. Green, this series, Vol. 18A, p. 414; R. Wei, this series, Vol. 18A, p. 424; S. G. Korenman and B. W. O'MaUey, this series, Voi. 18A, p. 427. a In our experience 30-50% of the radioactivity in commercially prepared [~4C~oiotin will not bind to avidin. The specific activity of samples diluted with unlabeled biotin must be adjusted to compensate for these impurities. 4 G. A. Bray, Anal. Biochem. 1, 279 (1960).
METHODS IN ENZYMOLOGY. VOL. 62
Copyright© 1979by AcademicPress.Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181962-0
[56]
EGG YOLK BIOTIN-BINDING PROTEIN
317
Purification Procedure 5
General. All buffers used are potassium phosphate, pH 7.2, at the concentration given. The procedure outlined is for 5 dozen eggs. Lipid Removal. e The eggs are cracked and the white and yolks are separated. The yolks are broken and 1.5 times the yolk volume in distilled water is added. The diluted yolk is well stirred. This solution is centrifuged at 13,200 g for 30 min. The supernatant is collected, and solid NaCl is added to make the solution 1 M in salt. After the salt has completely dissolved, the solution is poured into centrifuge bottles, two-thirds the solution volume in 1-butanol is added, and the mixture is shaken vigorously.r Centrifugation of the resulting emulsion at 13,200 g for 15 min causes a lipid-free aqueous phase and a lipid-rich organic phase to form, with a solid layer of precipitated protein at the interface. The two liquids are poured into a separatory funnel and the lower aqueous phase is removed. The aqueous phase is dialyzed overnight against running water at 4° to remove NaC1 and butanol. Phosphocellulose Ion-Exchange Chromatography. To the dialyzed lipid-free yolk solution is added 150 ml of settled phosphocellulose, equilibrated with 50 mM potassium phosphate, pH 7.2, and the suspension is stirred. The phosphocellulose is allowed to settle, and the supernatant is decanted. The phosphocellulose is poured into a column (2.5 × 50 cm) attached to a peristaltic pump and a UV column monitor. The column is washed with 50 mM potassium phosphate until the ODzs0 of the eluate has regained baseline. The flow is discontinued, and the 50 mM buffer is replaced by 0.5 M potassium phosphate. The pump is started, and the sharp peak of protein eluted is collected and pooled. Affinity Chromatography. The phosphocellulose eluate is placed in a pressure concentrator equipped with an ultrafiltering membrane (0.45 /zm) along with l0 ml of biotinyl-Sepharose, s The total volume must not exceed 150 ml in a 400-ml concentrator. The concentrator is lowered into a 55 ° water bath equipped for magnetic stirring° and pressurized with nitrogen, taking care not to increase the volume in the concentrator. The af5 H. W. Meslar, S. A. Camper, and H. B. White, III, J. Biol. Chem. 253, 6979 (1978). 6 H. W. Meslar and H. B. White, III, Anal. Biochem. 91, 75 (1978). 7 R. K. Morton, this series, Vol. l, p. 40. a Biotinyl-Sepharose is prepared by water-soluble carbodiimide coupling of biotin to hexamethylene-diamine substituted Sepharose 4BCL. The coupling must take place at pH 5.7-6.0. Below pH 5.7 the biotin becomes insoluble, and pH 6.0 is the upper limit for successful carbodiimide coupling. See I. Parikh, S. March, and P. Cuatrecasas, this series, Vol. 34B, p. 77. a An aluminum pot on top of a heater-stirrer has been used quite successfully in our laboratory for this purpose.
318
BIOTIN AND DERIVATIVES
[56]
SUMMARY OF PURIFICATION PROCEDURE
Fraction
Total units
Specific activity (units/mg)a
Diluted yolk Phosphocellulose eluate Gel filtration eluate b
378 58.6 5.25
6.82 × 10-3 1.42 12-13
a Protein determinations were made using the Bio-Rad dye binding assay kit, Bio-Rad Laboratories, Richmond, California. b Activity of the affinity gel eluate is not measured because of the excess unlabeled biotin present at that stage.
finity gel-protein slurry is washed with 2.0-2.3 liters of 50 mM potassium phosphate and depressurized. The gel is poured into a water-jacketed column (1.0 × 15 cm) and washed with 0.3 M potassium phosphate until the OD~0 of the eluate reaches baseline. The water jacket is filled with water at 65 °, and the column is filled with 10 ml of 40 mM biotin, dissolved in 0.2 M potassium phosphate. The system is allowed to heat and then is slowly eluted (0.6 ml/min). The entire biotin-containing peak is pooled. Gel Filtration. The eluted material is concentrated to a volume of 2 ml, made dense with solid sucrose, and applied to a column of Ultrogel AcA 44, l° equilibrated with 0.2 M potassium phosphate, and eluted. Fractions of constant specific activity are pooled and lyophilized. Summary of the Purification. The procedure is summarized in the table. This procedure has been successfully repeated 10 times. The protein shows a single band on gel electrophoresis, shows a single N-terminal, and gives a single precipitin band with antiserum raised against the mixture of proteins eluted from phosphocellulose. Properties
Stability. The protein is unstable in dilute solution at low ionic strength (50 mM potassium phosphate). At higher ionic strength, it is stable for months when frozen. The protein denatures at temperatures above 70° . Molecular Structure. Egg yolk biotin-binding protein appears to be a tetramer of identical subunits. The molecular weight of the tetramer is about 74,300. pH Optimum. Biotin is tightly bound from pH 5 to above pH 10. Below pH 5.0, the biotin-binding activity decreases and at pH 2.3 the protein is completely inactive. ~o LKB Instruments, RockviUe, Maryland.
[571
BIOTIN-BINDING ANTIBODIES
3 19
Temperature-Induced Biotin Release. Yolk biotin-binding protein has a maximum rate of biotin release at 70 °, above which temperature the protein begins to denature. Comparison with Egg White Avidin. 5 The properties of yolk biotinbinding protein are very different than those reported for avidin. 11 The pI is about pH 4.6, as opposed to about pH 10 for avidin. The yolk protein, although heavier (74,300 as opposed to 68,000), contains fewer residues of lysine, threonine, isoleucine, and aspartic acid. Biotin release is more than 1000-fold faster from the egg yolk protein than from the egg white protein. Neither protein cross-reacts with antibodies directed against the other. Cyanogen bromide cleavage generates peptides containing Nterminal leucine from egg yolk biotin-binding protein and N-terminal threonine and tryptophan from avidin. 11 N. M. Green, Adv. Protein Chem. 29, 85 (1975).
[57] A n t i b o d i e s T h a t B i n d B i o t i n a n d Inhibit Biotin-Containing Enzymes a By M E L V I N BERGER
Antibodies with affinity for biotin are produced following immunization of rabbits with antigens consisting of biotin linked covalently as a hapten to a carrier protein such as bovine serum albumin or bovine y-globulin, z In recent years considerable technology has been developed for the use of specific antibodies as biochemical reagents, a'4 Antibodies that bind biotin thus could be very useful in the purification and analysis of biotin-containing enzymes. In particular, the Fab fragments of these antibodies could be used as specific, monovalent, biotin-binding proteins, offering an advantage over the tetravalent avidin molecule in some cases. The avidity of these antibodies for enzyme-bound biotin allows enzyme inhibition to be used as a simple assay for the production of biotin-binding antibody. Enzyme inhibition can also serve as a convenient measure of unsaturated binding sites in studies of the interaction of biotin or its derivatives with the antibodies. I This work was supported by National Institutes of Health Grants Nos. 5T01-GM-00035 and AM-12245 and was performed in the laboratory of Dr. Harland G. Wood in the Department of Biochemistry, Case Western Reserve University. 2 M. Berger, Biochemistry 14, 2338 (1975). 3 j. B. Robbins and R. Schneerson, this series, Vol. 34, p. 703. 4 D. M. Livingston this series, Vol. 34, p. 723.
METHODS IN ENZYMOLOGY, VOL. 62
Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181962-0