[51]
F L U O R O M E T R I C ASSAY F O R A V I D I N A N D B I O T I N
287
In case of material having a lower biotin content, a larger amount of the starting material is used. This is followed by concentration of the biotin in the hydrolyzate. Up to 10 g of the original material can be used and the filtered hydrolyzate diluted to 100 ml with water. To this 1 g of activated charcoal (Norit A) is added. The mixture is shaken for 2 hr at room temperature and centrifuged. The pelleted charcoal is washed by resuspension in 8 ml of water, mixed for 30 min, and centrifuged. The washed pellet is extracted sequentially with six 2-ml portions of a mixture of ethanol-water-ammonia (10: 10: 1). The extracts are pooled and the biotin content is determined. Limit of Determination. This method can be applied to the determination of biotin in the range 0-1.0 ng per assay tube, which is comparable to the range of the microbiological assay with Lactobacillus plantarum ATCC 8014. Application. The isotope dilution procedure is sensitive enough to be used for the assay of biotin in biological material such as tissues and serum,
[51] A R a p i d ,
Sensitive Fluorometric Avidin and Biotin
Assay for
B y H E N R Y J . L I N a n d JACK F . K I R S C H
Principle. The binding of biotin to avidin is accompanied by a decrease in the fluorescence intensity of the protein. 1,2 These observations, combined with the known high affinity of avidin for biotin, led to the development of the assay described. Although not designed for biological fluids containing fluorescent impurities, the present method provides the most rapid and sensitive assay for samples containing relatively pure material. It is particularly convenient in that commercial avidin and biotin can be used directly. No radioisotopic or chemical syntheses are required, a Reagents Sodium phosphate buffer, 0.15 M, pH 7.0 Biotin standard (Sigma), ca. 20/ag/ml (80/zM) Avidin standard (Worthington), ca. 250 gg/ml (3.7/d~/) Procedure. All solutions are prepared in 0.15 M sodium phosphate buffer at pH 7, ionic strength = 0.35. The fluorescence measurements 1 N. M. Green, Biochem J. 90, 564 (1964). C. F. Chignell, D. K. Starkweather, and B. K. Sinha, J. Biol. Chem. 250, 5622 (1975). a H. J. Lin and J. F. Kirsch, Anal. Biochem. 81, 442 (1977).
METHODS IN ENZYMOLOGY. VOL. 62
Copyright © 1979by Academic Press, Inc. All rights of reproductionin any formreserved. ISBN 0-12-181962-0
288
BIOTIN AND DERIVATIVES I00
i
[51]
i
9O
g
80
o ~-
70
g: 60
50
I
I
I0
15
Biotin/avidin (/.~g/rag)
FIG. 1. Typical titration of avidin with biotin. About 25 p.g of avidin in 2.0 ml (180 nM) of 0.15 M sodium phosphate buffer, pH 7, was titrated with 2.0-/zl aliquots of a 90/zM (22 /zg/ml) solution of biotin. Fluorescence was measured at 350 nm with excitation at 290 nm. Each avidin molecule is tetrameric and binds four molecules of biotin IN. M. Green, Biochem. J. 92, 16C (1964)]. Data ofH. J. Lin and J. F. Kitsch,Anal. Biochem. 81,442 (1977).
described were made with a Hitachi Perkin-Elmer spectrofluorometer (Model MPF-2A) whose emission monochromator was calibrated with indole (Aldrich Chemical Company, m.p. 52-530). 4 All measurements were made in the ratio recording mode with the temperature between 20 and 24 °, the excitation monochromator set at 290 nm using a bandwidth of 4 nm, and the fluorescence monitored after each addition of titrant at 350 nm with a bandwidth of 16 nm. Assay of Avidin. A standard solution of biotin is prepared by dissolving 5 mg in 5.0 ml of buffer followed by dilution to 20/zg/ml. Two milliliters of approximately 10/~g/ml of avidin solution are pipetted into a 4-ml fluorometer cell and titrated with successive 2.0-/zl aliquots of the standard 20-/~g/ml biotin solution using a syringe microburete (e.g., Micrometric Instrument Co., Model SB2). Additions are made until no further decrease in fluorescence is observed, indicating the attainment of the equivalence point (Fig. 1). Assay of Biotin. Typically, a solution of avidin standardized with biotin as described above is diluted to 250/~g/ml and is used to titrate 2 ml of a solution that contains an unknown amount of biotin in the fluorometer cell. Relative fluorescence is measured after each addition of 2.0-/zl aliquots of the avidin solution from a ,syringe microburette, and a plot is made of fluorescence versus volume of avidin solution. The point at which the slope undergoes a sharp increase indicates the equivalence 4 I. B. Berlman, "Handbook of Fluorescence Spectra of Aromatic Molecules," 1st ed., p. 101. Academic Press, New York, 1965.
[51]
FLUOROMETRIC ASSAY FOR AVID1N AND BIOTIN
289
~o0
6O ~ 40, Is.
o
4~)
20
30
Volume Avidin Solution Added [H-J]
FIG. 2. Typical titration of biotin and avidin. Biotin, 33 ng in 2.0 ml (68 riM) of 0.15 M sodium phosphate buffer, pH 7, was titrated with 2.0-t,d aliquots of a solution of 250 gg of avidin (3.7 tB/) per milliliter. Fluorescence was measured at 350 nm with excitation at 290 nm. Data of H. J. Lin and J. F. Kirsch Anal. Biochem. $1,442 (1977).
point, and the quantity of biotin is determined from the specific activity of the avidin solution (Fig. 2). Notes. The useful range of the biotin assay is 20 to 120 ng. Between these limits, dilution upon addition of avidin will be less than 5%. Use of a wider emission slit may improve sensitivity. Ionic strength is not critical, since identical results have been obtained with 0.25 M sodium phosphate buffer, in which ionic strength = 0.6? The general applicability of these assays may be limited by the need to obtain samples nearly free of other fluorescent species, such as protein and free tryptophan. To achieve maximum sensitivity, the tryptophan concentration, for example, should not greatly exceed 1 - 2 / A / . A few other proteins are known to bind biotin, such as streptavidin 5 and a recently described egg yolk protein, ~ but avidin is the only biotin-binding protein that has been found in egg white. For samples from which interfering substances are absent, the assay is rapid and extremely parsimonious, since only microgram quantities of avidin are required to bind 20 ng of biotin.
L. Chalet and F. J. Wolf, Arch. Biochem. Biophys. 106, 1 (1964). e H. G. White, B. A. Dcnnison, M. A. Della Fera, C. J. Whitney, J. C. McGuire, H. W. Meslar, and P. H. Sammelwitz, Biochem. J. 157, 395 (1976).
F L U O R O M E T R I C ASSAY F O R A V I D I N A N D B I O T I N
287
In case of material having a lower biotin content, a larger amount of the starting material is used. This is followed by concentration of the biotin in the hydrolyzate. Up to 10 g of the original material can be used and the filtered hydrolyzate diluted to 100 ml with water. To this 1 g of activated charcoal (Norit A) is added. The mixture is shaken for 2 hr at room temperature and centrifuged. The pelleted charcoal is washed by resuspension in 8 ml of water, mixed for 30 min, and centrifuged. The washed pellet is extracted sequentially with six 2-ml portions of a mixture of ethanol-water-ammonia (10: 10: 1). The extracts are pooled and the biotin content is determined. Limit of Determination. This method can be applied to the determination of biotin in the range 0-1.0 ng per assay tube, which is comparable to the range of the microbiological assay with Lactobacillus plantarum ATCC 8014. Application. The isotope dilution procedure is sensitive enough to be used for the assay of biotin in biological material such as tissues and serum,
[51] A R a p i d ,
Sensitive Fluorometric Avidin and Biotin
Assay for
B y H E N R Y J . L I N a n d JACK F . K I R S C H
Principle. The binding of biotin to avidin is accompanied by a decrease in the fluorescence intensity of the protein. 1,2 These observations, combined with the known high affinity of avidin for biotin, led to the development of the assay described. Although not designed for biological fluids containing fluorescent impurities, the present method provides the most rapid and sensitive assay for samples containing relatively pure material. It is particularly convenient in that commercial avidin and biotin can be used directly. No radioisotopic or chemical syntheses are required, a Reagents Sodium phosphate buffer, 0.15 M, pH 7.0 Biotin standard (Sigma), ca. 20/ag/ml (80/zM) Avidin standard (Worthington), ca. 250 gg/ml (3.7/d~/) Procedure. All solutions are prepared in 0.15 M sodium phosphate buffer at pH 7, ionic strength = 0.35. The fluorescence measurements 1 N. M. Green, Biochem J. 90, 564 (1964). C. F. Chignell, D. K. Starkweather, and B. K. Sinha, J. Biol. Chem. 250, 5622 (1975). a H. J. Lin and J. F. Kirsch, Anal. Biochem. 81, 442 (1977).
METHODS IN ENZYMOLOGY. VOL. 62
Copyright © 1979by Academic Press, Inc. All rights of reproductionin any formreserved. ISBN 0-12-181962-0
288
BIOTIN AND DERIVATIVES I00
i
[51]
i
9O
g
80
o ~-
70
g: 60
50
I
I
I0
15
Biotin/avidin (/.~g/rag)
FIG. 1. Typical titration of avidin with biotin. About 25 p.g of avidin in 2.0 ml (180 nM) of 0.15 M sodium phosphate buffer, pH 7, was titrated with 2.0-/zl aliquots of a 90/zM (22 /zg/ml) solution of biotin. Fluorescence was measured at 350 nm with excitation at 290 nm. Each avidin molecule is tetrameric and binds four molecules of biotin IN. M. Green, Biochem. J. 92, 16C (1964)]. Data ofH. J. Lin and J. F. Kitsch,Anal. Biochem. 81,442 (1977).
described were made with a Hitachi Perkin-Elmer spectrofluorometer (Model MPF-2A) whose emission monochromator was calibrated with indole (Aldrich Chemical Company, m.p. 52-530). 4 All measurements were made in the ratio recording mode with the temperature between 20 and 24 °, the excitation monochromator set at 290 nm using a bandwidth of 4 nm, and the fluorescence monitored after each addition of titrant at 350 nm with a bandwidth of 16 nm. Assay of Avidin. A standard solution of biotin is prepared by dissolving 5 mg in 5.0 ml of buffer followed by dilution to 20/zg/ml. Two milliliters of approximately 10/~g/ml of avidin solution are pipetted into a 4-ml fluorometer cell and titrated with successive 2.0-/zl aliquots of the standard 20-/~g/ml biotin solution using a syringe microburete (e.g., Micrometric Instrument Co., Model SB2). Additions are made until no further decrease in fluorescence is observed, indicating the attainment of the equivalence point (Fig. 1). Assay of Biotin. Typically, a solution of avidin standardized with biotin as described above is diluted to 250/~g/ml and is used to titrate 2 ml of a solution that contains an unknown amount of biotin in the fluorometer cell. Relative fluorescence is measured after each addition of 2.0-/zl aliquots of the avidin solution from a ,syringe microburette, and a plot is made of fluorescence versus volume of avidin solution. The point at which the slope undergoes a sharp increase indicates the equivalence 4 I. B. Berlman, "Handbook of Fluorescence Spectra of Aromatic Molecules," 1st ed., p. 101. Academic Press, New York, 1965.
[51]
FLUOROMETRIC ASSAY FOR AVID1N AND BIOTIN
289
~o0
6O ~ 40, Is.
o
4~)
20
30
Volume Avidin Solution Added [H-J]
FIG. 2. Typical titration of biotin and avidin. Biotin, 33 ng in 2.0 ml (68 riM) of 0.15 M sodium phosphate buffer, pH 7, was titrated with 2.0-t,d aliquots of a solution of 250 gg of avidin (3.7 tB/) per milliliter. Fluorescence was measured at 350 nm with excitation at 290 nm. Data of H. J. Lin and J. F. Kirsch Anal. Biochem. $1,442 (1977).
point, and the quantity of biotin is determined from the specific activity of the avidin solution (Fig. 2). Notes. The useful range of the biotin assay is 20 to 120 ng. Between these limits, dilution upon addition of avidin will be less than 5%. Use of a wider emission slit may improve sensitivity. Ionic strength is not critical, since identical results have been obtained with 0.25 M sodium phosphate buffer, in which ionic strength = 0.6? The general applicability of these assays may be limited by the need to obtain samples nearly free of other fluorescent species, such as protein and free tryptophan. To achieve maximum sensitivity, the tryptophan concentration, for example, should not greatly exceed 1 - 2 / A / . A few other proteins are known to bind biotin, such as streptavidin 5 and a recently described egg yolk protein, ~ but avidin is the only biotin-binding protein that has been found in egg white. For samples from which interfering substances are absent, the assay is rapid and extremely parsimonious, since only microgram quantities of avidin are required to bind 20 ng of biotin.
L. Chalet and F. J. Wolf, Arch. Biochem. Biophys. 106, 1 (1964). e H. G. White, B. A. Dcnnison, M. A. Della Fera, C. J. Whitney, J. C. McGuire, H. W. Meslar, and P. H. Sammelwitz, Biochem. J. 157, 395 (1976).
