- Email: [email protected]
[76] Newer methods of avidin assay
[76]
NEWER METHODS OF AVIDIN ASSAY
427
solution further). Add 1 ~1 of the biotin solution to the sample cuvette, mix, and record the increase in absorbancy at 233 m~. Continue to add 1-~1 aliquots of biotin until no further increase in absorbancy is observed. Plot the results on graph paper to determine the equivalence point. The specific activity can then be calculated by units/rag =
(tA biotin used at equivalence point)(cone, in #g/ul) mg avidin per 3 ml
[76] Newer Methods
of Avidin Assay
B y S. G. KORENMAN, and B. W. O'MALLEY
Recent renewed interest in avidin, the biotin binding protein of egg white, has been based both on its unusual chemical properties and on hormonal control of its synthesis by the chick oviduct. Study of the protein required assay procedures less cumbersome and more precise than the microbiological technique of Eakin, Snell, and Williams? Melamed and Green s devised a method of preparing relatively pure avidin. Using this material, Green determined the molecular weight and number of binding sites, 3 the kinetics of the binding, ~ and the nature of the binding site. ~ During the course of these studies, two spectrophotometric assays 6,7 and a radioligand binding assay 8 for avidin were used. Because of our interest in the mechanisms of steroid hormone action, we sought a model system in which the synthesis of specific proteins is under the control of individual hormones. I t was apparent that regulation of egg-white protein synthesis by sex hormones represents such a system, and particularly t h a t avidin biosynthesis, which is regulated by both estrogens and progesterone, 9 would be of great interest. Accordingly, a convenient, specific, rapid, and precise radioligand binding assay system for avidin suitable for the study of tissue fractions has been developed. TM B y means of 1R. E. Eakin, E. E. Snell, and R. J. Williams, J. Biol. Chem. 140, 535 (1941). M. D. Melamed and N. M. Green, Biochem. J. 89, 591 (1963). 3N. M. Green, Biochem. J. 92, 16c (1964). N. M. Green, Biochem. J. 89, 585 (1963). 5N. M. Green, Biochem. J. 89, 599 (1963). See footnote 5. N. hi. Green, Biochem. J. 94, 23c (1965). 8N. M. Green, Biochem. J. 89, 599 (1963). ' R. M. Fraps, R. Hertz, and W. H. Sebrell, Proc. Soc. Exptl. Biol. Med. 52, 140 (1943). ~oS. G. Korenman and B. W. O'Malley, Biochim. Biophys. Acla 140, 174 (1967).
428
BIOTIN AND DERIVATIVES
[76]
this procedure, considerable information has been gathered on the mechanism of action of progesterone in stimulating avidin biosynthesisY -~a It has also been possible, emph)ying a specific antiserum, to develop an immunoassay for avidinJ 4
Avidin Standards Avidin of high specific activity is not always commercially available. Crude avidin preparations can be used as standards as long as their content of unsaturated biotin binding sitcs is carefully measured and their specific activity is determined. One unit of avidin activity is defined as that required to bind 1 t*g of biotin. Pure avidin contains 13.8 l : / m g ) 5 By the method of Melamed and Green, l~ employing ion-exchange chrom-ttography on carboxymethyl cellulose, material with a specific activity of 10 U / m g or greater can be prepared from egg whites. The ingenious procedure devised by McCormick ~7 using a biotin-celluh)se column is another means that can be used to enrich crude avidin preparations.
Spectrophotometric Assays A. Titration of "~vidin (~oncentration by measurement of the increase in optical density at 233 m~ produ('ed by addition of biotin until all the binding sites of avidin are saturated) s This assay is insensitive and depends upon the accuracy of the titration procedure. B. Binding of the anionic dye, 4'-hydroxyazobenzene-2-carboxylate, to avidin was shown to result in a new absorption band at 500 mr*)" The dye is bound at the same sites as biotin and can be displaced by biotin. For assay purposes, to ensure specificity, dye is added until the binding sites of a dilute avidin solution at ptI 7.0 are just :.~aturated, and then by titration the an~ount of biotin needed to eliminate the "tbsorbance at 500 mr* is determined. The method is sensitive to about 25 ug/ml of avidin. It is not suitable for assay of tissue fractions containing only a small amount of avidin because dye binding to many proteins may oc('ur and the effects of biotin titration may not be discernible. ~1S. G. Koremnan and B. W. O'Malley, E~ldocrinology 83, 11 (1968). 12B. W. O'Malley, Biochemislry 6, 2546 (1967). 13B. W. O'3,Ialley, S. L. McGuire, P. (). Kohler, and S. G. Korenman, Recent Progr. Hormone Res. 25, 105 (1969). 14B. W. O'Malley and S. G. Korenman, Life Sci. 6, 1953 (1967). 15M. D. Melamed and N. M. Green, Biochem. ~ 89, 591 (1963). 16See fooinole 15. 171). B. McCormick, Ann. Biochem. 13, 194 (1965). is N. M. Green, Biochem. J. 89, 599 (1(.)63). 19N. M. Green, Biochem. J. 94, 23c (1965).
[75]
NEWER METHODS OF AVIDIN ASSAY
429
Radioligand Binding Assays In general, these procedures involve interaction of a labeled ligand, such as biotin, with a specific binding protein, such as avidin. By measurement of the amount of radioactivity either in the ligand-protein complex or unbound, under the appropriate conditions, the amount of either the ligand or the protein can be determined. In these assays, to measure avidin content an excess of radioactive biotin is reacted with avidin, the avidin-biotin complex is separated from unbound biotin, and avidin is measured by the number of counts bound. The sensitivity of the method is limited only by the specific activity of the labeled biotin. In reality, the number of unsaturated biotin binding sites, rather than the total immber of avidin molecules present, are measured. Because of the extremely low equilibrium constant of dissociation of the avidin-biotin complex, 20 the binding reaction may be considered irreversible. Green used both batch adsorption to carboxymethyl cellulose and ultrafiltration to separate bound from free biotin, 2~ while Wei and Wright employed gel filtration on Sephadex. 22 Korenman and O'Malley 2a used adsorption of the avidin-biotin complex onto bentonite. Only the latter procedure, which is the most convenient, will be described in detail. Water is distilled and deionized to ensure that it is iron free. Bentonite is used as obtained. Cellulose nitrate filters, 0.45-~ pore size, 25 mm in diameter, are obtained from the Millipore Corporation. Biotin 14C with a specific activity of 50 uCi/mg is used. All of a sample must be shown tobind to an excess of avidin (radioactive biotin should always be tested for purity by demonstrating that it binds quantitatively to avidin). Samples and standards are assayed in triplicate. Reagents and avidin samples are diluted in 0.2 M amm(._',ium carbonate. The avidin and biotin solution are mixed, taken to 0.6 ml, and incubated for 10 minutes at 23 °. One milliliter of 0.2 M ammonium carbonate containing 10 mg of bentonite is then added. The suspension is stirred, incubated for 5 minutes, and transferred to a Millipore filter under suction and rinsed once. Unbound biotin passes through the filter. The filter is transferred to a counting vial, wet with 1 drop of ethanol, and dissolved in Bray's solution, "-4leaving a bentonite sediment. Radioactivity is measured in a liquid scintillation spectrometer at high efficiency. Using the available biotin specific activity, avidin coneentrations as low as 0.1 ~,g/ml can be measured with ease. The sensitivity 2oN. M. Green, Biochem. J. 89, 585 (1963). 21See footnote 20. 52R. D. Wei and L. D. Wright, Proc. Soc. Exptl. Biol. Med. 117, 17 (1964). 23S. G. Korenman and B. W. O'Malley, Biochim. Biophys. Acts 140, 174 (1967). 24G. A. Bray, Ann. Biochem. 1, 279 (1960).
430
BIOTIN AND DERIVATIVES
[76]
can be increased by utilizing tritiated biotin if it is available. This procedure is suitable for avidin assay in crude oviduct supernatant fractions. Immunoassay
Avidin is quite antigenic?~ By the use of rabbit antiavidin and sheep antirabbit ~,-globulin, O'Malley and Korenman ~6 were able both to assay avidin by biotin 14C binding and to determine that portion which was newly synthesized by simultaneous measurement of incorporation of a radioactive amino acid into antibody-precipitable protein. Specific antibodies are produced in rabbits by giving 4 weekly injections of avidin in Freund's adjuvant and then bleeding the animals. Sheep antirabbit ~,-globulin is obtained commercially. The reactions are carried out at 23 °. Avidin is incubated with a small excess of specific antiserum for 2 hours. Then an excess of biotin 14C is added and incubation is continued for 20 minutes. The avidin-antibody complex binds biotin just as well as free avidin. An excess of sheep antirabbit -r-globulin is added, and incubation is carried out for 3 hours. The resultant precipitate is centrifuged, washed, dissolved in 1 ml of NCS reagent (Nuclear Chicago Corp.), added to 10 ml of toluene phosphor, and counted in a liquid scintillation spectrometer. If the avidin specifically precipitated has been newly formed in the presence of a tritiated amino acid (i.e., as a result of induction by progesterone) the proportion of new protein synthesis due to avidin and the specific activity of the avidin can be determined by simultaneous measurement of 14C and 3H radioactivity. Availability of radioactively labeled biotin has made it possible to develop a number of convenient assay procedures for avidin suitable for study of both its biochemical characteristics and regulation of its biosynthesis. The procedures outlined can be modified with ease to permit quantitative assay of streptavidin, biotin, and actively binding biotin analogs as well.
26 D. V. Siva Sankar, B. J. Cossano, H. W. Theis, and C. R. Marks, Nature 181, 619 (1958). 26 B. W. O'Malley and S. G. Korenman, Life Sci. 6, 1953 (1967).
NEWER METHODS OF AVIDIN ASSAY
427
solution further). Add 1 ~1 of the biotin solution to the sample cuvette, mix, and record the increase in absorbancy at 233 m~. Continue to add 1-~1 aliquots of biotin until no further increase in absorbancy is observed. Plot the results on graph paper to determine the equivalence point. The specific activity can then be calculated by units/rag =
(tA biotin used at equivalence point)(cone, in #g/ul) mg avidin per 3 ml
[76] Newer Methods
of Avidin Assay
B y S. G. KORENMAN, and B. W. O'MALLEY
Recent renewed interest in avidin, the biotin binding protein of egg white, has been based both on its unusual chemical properties and on hormonal control of its synthesis by the chick oviduct. Study of the protein required assay procedures less cumbersome and more precise than the microbiological technique of Eakin, Snell, and Williams? Melamed and Green s devised a method of preparing relatively pure avidin. Using this material, Green determined the molecular weight and number of binding sites, 3 the kinetics of the binding, ~ and the nature of the binding site. ~ During the course of these studies, two spectrophotometric assays 6,7 and a radioligand binding assay 8 for avidin were used. Because of our interest in the mechanisms of steroid hormone action, we sought a model system in which the synthesis of specific proteins is under the control of individual hormones. I t was apparent that regulation of egg-white protein synthesis by sex hormones represents such a system, and particularly t h a t avidin biosynthesis, which is regulated by both estrogens and progesterone, 9 would be of great interest. Accordingly, a convenient, specific, rapid, and precise radioligand binding assay system for avidin suitable for the study of tissue fractions has been developed. TM B y means of 1R. E. Eakin, E. E. Snell, and R. J. Williams, J. Biol. Chem. 140, 535 (1941). M. D. Melamed and N. M. Green, Biochem. J. 89, 591 (1963). 3N. M. Green, Biochem. J. 92, 16c (1964). N. M. Green, Biochem. J. 89, 585 (1963). 5N. M. Green, Biochem. J. 89, 599 (1963). See footnote 5. N. hi. Green, Biochem. J. 94, 23c (1965). 8N. M. Green, Biochem. J. 89, 599 (1963). ' R. M. Fraps, R. Hertz, and W. H. Sebrell, Proc. Soc. Exptl. Biol. Med. 52, 140 (1943). ~oS. G. Korenman and B. W. O'Malley, Biochim. Biophys. Acla 140, 174 (1967).
428
BIOTIN AND DERIVATIVES
[76]
this procedure, considerable information has been gathered on the mechanism of action of progesterone in stimulating avidin biosynthesisY -~a It has also been possible, emph)ying a specific antiserum, to develop an immunoassay for avidinJ 4
Avidin Standards Avidin of high specific activity is not always commercially available. Crude avidin preparations can be used as standards as long as their content of unsaturated biotin binding sitcs is carefully measured and their specific activity is determined. One unit of avidin activity is defined as that required to bind 1 t*g of biotin. Pure avidin contains 13.8 l : / m g ) 5 By the method of Melamed and Green, l~ employing ion-exchange chrom-ttography on carboxymethyl cellulose, material with a specific activity of 10 U / m g or greater can be prepared from egg whites. The ingenious procedure devised by McCormick ~7 using a biotin-celluh)se column is another means that can be used to enrich crude avidin preparations.
Spectrophotometric Assays A. Titration of "~vidin (~oncentration by measurement of the increase in optical density at 233 m~ produ('ed by addition of biotin until all the binding sites of avidin are saturated) s This assay is insensitive and depends upon the accuracy of the titration procedure. B. Binding of the anionic dye, 4'-hydroxyazobenzene-2-carboxylate, to avidin was shown to result in a new absorption band at 500 mr*)" The dye is bound at the same sites as biotin and can be displaced by biotin. For assay purposes, to ensure specificity, dye is added until the binding sites of a dilute avidin solution at ptI 7.0 are just :.~aturated, and then by titration the an~ount of biotin needed to eliminate the "tbsorbance at 500 mr* is determined. The method is sensitive to about 25 ug/ml of avidin. It is not suitable for assay of tissue fractions containing only a small amount of avidin because dye binding to many proteins may oc('ur and the effects of biotin titration may not be discernible. ~1S. G. Koremnan and B. W. O'Malley, E~ldocrinology 83, 11 (1968). 12B. W. O'Malley, Biochemislry 6, 2546 (1967). 13B. W. O'3,Ialley, S. L. McGuire, P. (). Kohler, and S. G. Korenman, Recent Progr. Hormone Res. 25, 105 (1969). 14B. W. O'Malley and S. G. Korenman, Life Sci. 6, 1953 (1967). 15M. D. Melamed and N. M. Green, Biochem. ~ 89, 591 (1963). 16See fooinole 15. 171). B. McCormick, Ann. Biochem. 13, 194 (1965). is N. M. Green, Biochem. J. 89, 599 (1(.)63). 19N. M. Green, Biochem. J. 94, 23c (1965).
[75]
NEWER METHODS OF AVIDIN ASSAY
429
Radioligand Binding Assays In general, these procedures involve interaction of a labeled ligand, such as biotin, with a specific binding protein, such as avidin. By measurement of the amount of radioactivity either in the ligand-protein complex or unbound, under the appropriate conditions, the amount of either the ligand or the protein can be determined. In these assays, to measure avidin content an excess of radioactive biotin is reacted with avidin, the avidin-biotin complex is separated from unbound biotin, and avidin is measured by the number of counts bound. The sensitivity of the method is limited only by the specific activity of the labeled biotin. In reality, the number of unsaturated biotin binding sites, rather than the total immber of avidin molecules present, are measured. Because of the extremely low equilibrium constant of dissociation of the avidin-biotin complex, 20 the binding reaction may be considered irreversible. Green used both batch adsorption to carboxymethyl cellulose and ultrafiltration to separate bound from free biotin, 2~ while Wei and Wright employed gel filtration on Sephadex. 22 Korenman and O'Malley 2a used adsorption of the avidin-biotin complex onto bentonite. Only the latter procedure, which is the most convenient, will be described in detail. Water is distilled and deionized to ensure that it is iron free. Bentonite is used as obtained. Cellulose nitrate filters, 0.45-~ pore size, 25 mm in diameter, are obtained from the Millipore Corporation. Biotin 14C with a specific activity of 50 uCi/mg is used. All of a sample must be shown tobind to an excess of avidin (radioactive biotin should always be tested for purity by demonstrating that it binds quantitatively to avidin). Samples and standards are assayed in triplicate. Reagents and avidin samples are diluted in 0.2 M amm(._',ium carbonate. The avidin and biotin solution are mixed, taken to 0.6 ml, and incubated for 10 minutes at 23 °. One milliliter of 0.2 M ammonium carbonate containing 10 mg of bentonite is then added. The suspension is stirred, incubated for 5 minutes, and transferred to a Millipore filter under suction and rinsed once. Unbound biotin passes through the filter. The filter is transferred to a counting vial, wet with 1 drop of ethanol, and dissolved in Bray's solution, "-4leaving a bentonite sediment. Radioactivity is measured in a liquid scintillation spectrometer at high efficiency. Using the available biotin specific activity, avidin coneentrations as low as 0.1 ~,g/ml can be measured with ease. The sensitivity 2oN. M. Green, Biochem. J. 89, 585 (1963). 21See footnote 20. 52R. D. Wei and L. D. Wright, Proc. Soc. Exptl. Biol. Med. 117, 17 (1964). 23S. G. Korenman and B. W. O'Malley, Biochim. Biophys. Acts 140, 174 (1967). 24G. A. Bray, Ann. Biochem. 1, 279 (1960).
430
BIOTIN AND DERIVATIVES
[76]
can be increased by utilizing tritiated biotin if it is available. This procedure is suitable for avidin assay in crude oviduct supernatant fractions. Immunoassay
Avidin is quite antigenic?~ By the use of rabbit antiavidin and sheep antirabbit ~,-globulin, O'Malley and Korenman ~6 were able both to assay avidin by biotin 14C binding and to determine that portion which was newly synthesized by simultaneous measurement of incorporation of a radioactive amino acid into antibody-precipitable protein. Specific antibodies are produced in rabbits by giving 4 weekly injections of avidin in Freund's adjuvant and then bleeding the animals. Sheep antirabbit ~,-globulin is obtained commercially. The reactions are carried out at 23 °. Avidin is incubated with a small excess of specific antiserum for 2 hours. Then an excess of biotin 14C is added and incubation is continued for 20 minutes. The avidin-antibody complex binds biotin just as well as free avidin. An excess of sheep antirabbit -r-globulin is added, and incubation is carried out for 3 hours. The resultant precipitate is centrifuged, washed, dissolved in 1 ml of NCS reagent (Nuclear Chicago Corp.), added to 10 ml of toluene phosphor, and counted in a liquid scintillation spectrometer. If the avidin specifically precipitated has been newly formed in the presence of a tritiated amino acid (i.e., as a result of induction by progesterone) the proportion of new protein synthesis due to avidin and the specific activity of the avidin can be determined by simultaneous measurement of 14C and 3H radioactivity. Availability of radioactively labeled biotin has made it possible to develop a number of convenient assay procedures for avidin suitable for study of both its biochemical characteristics and regulation of its biosynthesis. The procedures outlined can be modified with ease to permit quantitative assay of streptavidin, biotin, and actively binding biotin analogs as well.
26 D. V. Siva Sankar, B. J. Cossano, H. W. Theis, and C. R. Marks, Nature 181, 619 (1958). 26 B. W. O'Malley and S. G. Korenman, Life Sci. 6, 1953 (1967).