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A sensitive fluorometric assay for avidin and biotin
ANALYTICAL
BIOCHEMISTRY
A Sensitive
81, 442-446 (1977)
Fluorometric
Assay for Avidin
and Biotin
HENRY J. LIN ANDJACK F. KIRSCH Department
of Biochemistry,
University
of California,
Berkeley,
California
WYtJ
Received December 20, 1976: accepted April 25. 1977 The quenching of the tryptophan fluorescence of avidin by biotin forms the basis for a rapid, sensitive assay for biotin and avidin in solutions free of fluorescent contaminants. Avidin solutions are assayed by titration with a standard biotin solution until no further decrease in fluorescence at 350 nm, with excitation at 290 nm. is observed. Unknown quantities of biotin in solution may be determined by titration with aliquots of standard avidin until the change in fluorescence observed following each addition undergoes a sharp increase. As little as 20 ng of biotin may be measured in this way.
Several methods for the assay of avidin, biotin, or both are currently available. The increase in the absorptivity of avidin at 233 nm upon the binding of biotin (AeZS3= 2.4 x IO4 M-' cm-’ mol-’ of biotin) (l-3) and the ability of biotin to displace quantitatively 4’-hydroxyazobenzene-2carboxylate from its colored complex with avidin (3,4) provide the bases for two assays for either the protein or biotin. The latter assay is sensitive to about 50 pg of avidin and 1 pug of biotin (3). More sensitive avidin assays have been accomplished using [14C]biotin (5-8) [lower limit = 20 ng of avidin (8)]. Biotin may be assayed chemically, using p -dimethylaminocinnamaldehyde (lower limit = 10 pg) (9,10), or with one of many biological procedures (11). Many organisms are suitable for the biological assays, such as Lactobacillusplantarum (12), Lactobacillus casei (13,14), Neurospora crassa (15), and Ochromonas danica (16) (typical lower limit = 0.01 ng), but these assays may take 3 to 5 days to perform. Green (17) has noted that the binding of biotin to avidin is accompanied by a decrease in the fluorescence intensity of the protein, an observation later confirmed by Chignell and co-workers (18). This suggests that, because of the high affinity of avidin for biotin, these observations may be generalized into an assay for either avidin or biotin. 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. A procedure for the determination of free biotin in amounts as small as 20 ng is described below. 442 Copyright P 1977 by Academr Press. Inc. All rights of reproduction m any form reserved
1SSN ooO3-2697
RAPID ASSAY FOR AVIDIN
AND BIOTIN
443
EXPERIMENTAL
Avidin and d-biotin [mp 229-230°C lit. mp 230-232°C (19)] were purchased from Worthington Biochemical Corp. and from Sigma, respectively. Fluorescence measurements were made with a Hitachi PerkinElmer spectrofluorometer (Model MPF-2A) whose emission monochromator had been calibrated with indole (Aldrich Chemical Company, mp 52-53°C) (20). All measurements were made in the ratio recording mode with the temperature between 20 and 24°C. All solutions were made in 0.15 M sodium phosphate buffer at pH 7, ionic strength = 0.35. 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 pg/ml. Two milliliters of approximately 10 pg/rnl of avidin solution is pipetted into a 4-ml fluorometer cell and titrated with successive 2.0-~1.1aliquots of the standard 20-p.g/ml biotin solution using a syringe microburet (Micrometric Instrument Co., Model SB2). The excitation monochromator is set at 290 nm using a bandwidth of 4 nm, and the fluorescence is monitored after each addition at 350 nm using a bandwidth of 16 nm. Additions are made until no further decrease in fluorescence is observed, indicating the attainment of the equivalence point (Fig. 1). Titration of biotin. Typically, a solution of avidin standardized with biotin as described above is diluted to 250 pg/ml and is used to titrate 2 ml of a solution that contains an unknown amount of biotin in a fluorometer
Biotin/Avidin
[pg/mg]
FIG. 1. Typical titration of avidin with biotin. About 25 fig of avidin in 2.0 ml of 0.15 M sodium phosphate buffer, pH 7, was titrated with 2.0-4 aliquots of a 90 pM (22 &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 (22).
444
LIN AND KIRSCH
0
20 Volume Avidin Solution Added [PI]
30
IO
FIG. 2. Typical titration of biotin with avidin. Biotin, 33 ng in 2.0 ml of 0.15 M sodium phosphate buffer, pH 7, was titrated with 2.0~~1 ahquots of a 250+FLg/ml avidin solution. Fluorescence was measured at 350 nm with excitation at 290 nm.
cell. Relative fluorescence is measured after each addition of 2.03~1 aliquots of the avidin solution from a syringe microburet, 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 point, and the quantity of biotin is determined from the specific activity of the avidin solution (Fig. 2). RESULTS AND DISCUSSION
The fluorescent properties of free avidin and of avidin saturated with biotin are summarized in Table 1. The results of titrating avidin with biotin and of titrating biotin with avidin, as described under Experimental, are illustrated in Figs. 1 and 2. TABLE FLUORESCENT
PROPERTIES
OF FREE
Emission hmaxb(nm) Relative fluorescence at 350 nm (bandwidth = 16 nm)
AVIDIN
1
AND
OF AVIDIN
SATURATED
WITH
BIOTIN”
Free avidin
Avidin saturated with biotin
342
332
100
55 2 0.8
u In 0.15 or 0.25 M Na-phosphate buffers, 20-24°C. with excitation (bandwidth = 4 nm). Avidin concentration = 13 pg/ml. * A shift from 336 to 326 nm is reported by Chignell et al. (18).
at 290 nm
RAPID
ASSAY
FOR
AVIDIN
AND
BIOTIN
445
The quenching of the protein fluorescence by biotin is consistent with the observation that avidin-bound biotin protects all four tryptophan residues of an avidin subunit from oxidation by N-bromosuccinimide (1,2,2 I-23). The avidin specific activities determined by titration of avidin with biotin (11 .O ? 0.2 pg of biotin/mg, seven determinations) and by titration of biotin with avidin (10.8 2 0.2 pg of biotin/mg, four determinations) agree well with that given by the manufacturer (10.6 pg of biotin/mg). 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 l-2 PM. [Biological fluids have high concentrations of tryptophan, e.g., 54 PM in human serum (24) and 69 PM in human urine (25), compared to their concentrations of biotin, about 1 nM in serum and 70 nM in urine (16)]. A few other proteins are known to bind biotin, such as streptavidin (26) and a recently described egg yolk protein (27). 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. ACKNOWLEDGMENT This work Foundation.
was
supported
by
a grant
(BMS
74-17633)
from
the
National
Science
REFERENCES I. Green. N. M. (1962) Biochim. Biophys. Acru 59, 244-246. 2. Green, N. M. (1963) Biochrm. J. 89, 599-609. 3. Green, N. M. (1970)in Methods in Enzymology (McCormick. D. B.. and Wright. L. D.. eds.). Vol. 18A. pp. 418-424. Academic Press, New York. 4. Green, N. M. (1965) Biochem. J. 94, 23C-24C. 5. Wei, R. D., and Wright. L. D. (1964) Proc. Sot. Exp. Biol. Med. 117, 17-20. 6. Wei. R. D. (1970) in Methods in Enzymology (McCormick, D. B., and Wright, L. D., eds.). Vol. 18A. pp. 424-427, Academic Press, New York. 7. Korenman. S. G., and O’Malley, B. W. (1967) Bio&‘m. Biophys. Ac-ru 140, 174-176. 8. Korenman. S. G., and O’Malley. B. W. (1970)in Methods in Enzymology (McCormick. D. B.. and Wright. L. D.. eds.). Vol. 18A, pp. 427-430, Academic Press. New York. 9. McCormick. D. B., and Roth, J. A. (1970) Anal. Biochem. 34, 226-236. IO. McCormick, D. B., and Roth, J. A. (1970) in Methods in Enzymology (McCormick, D. B., and Wright, L. D.. eds.). Vol. 18A, pp. 383-385, Academic Press, New York. 11. Gyiirgy. P., and Langer. B. W.. Jr. (1968) in The Vitamins (Sebrell, W. H., Jr.. and Harris, R. S.. eds.). 2nd ed., Vol. 2. pp. 2X0-284, Academic Press. New York.
446
LIN
AND
KIRSCH
12. Wright, L. D., and Skeggs. H. R. (1944) Proc. Sot. Exp. Biol. Med. 56, 95-98. 13. Shull, G. M.. Hutchings. B. L., and Peterson, W. H. (1942) .I. Bio/. Chem. 142, 913-920. 14. Shull. G. M.. and Peterson, W. H. (1943) J. Biol. Chem. 151, 201-202. 15. Hodson. A. Z. (1945) J. Biol. Chem. 157, 383-385. 16. Baker, H.. Frank, O., Matovitch, V. B.. Pasher, I., Aaronson. S., Hutner, S. H., and Sobotka, H. (1962) Anal. Biochem. 3, 31-39. 17. Green, N. M. (1964) Biochem. .I. 90, 564-568. 18. Chignell, C. F.. Starkweather, D. K.. and Sinha, B. K. (1975) J. Biol. Chem. 250, 5622-5630.
19. du Vigneaud. V., Hofmann, K., Melville, D. B., and Rachele, J. R. (1941) J. Bio/. Chem. 140, 763-766. 20. Berlman. I. B. (1%5) Handbook of Fluorescence Spectra of Aromatic Molecules, p. 101, Academic Press,New York. 21. Green, N. M., and Toms, E. J. (1972) Biochem. J. 130, 707-711. 22. Green, N. M. (1964) Biochem. J. 92, l6C-l7C. 23. DeLange, R. J., and Huang, T. S. (1971) J. Biol. Chem. 246, 698-709. 24. Hier. S. W.. and Bergeim. 0. (1946) J. Biol. Chem. 163, 129-135. 25. Woodson, H. W., Hier. S. W., Solomon, J. D., and Bergeim, 0. (1948) J. Biol. Chem. 172, 613-618. 26. Chaiet, L., and Wolf, F. J. (1964) Arch. Biochem. Biophys. 106, l-5. 27. White. H. B., Dennison, B. A.. Della Fera. M. A., Whitney, C. J., McGuire, J. C.. Meslar. H. W.. and Sammelwitz, P. H. (1976) Biochem. J. 157, 395-400.
BIOCHEMISTRY
A Sensitive
81, 442-446 (1977)
Fluorometric
Assay for Avidin
and Biotin
HENRY J. LIN ANDJACK F. KIRSCH Department
of Biochemistry,
University
of California,
Berkeley,
California
WYtJ
Received December 20, 1976: accepted April 25. 1977 The quenching of the tryptophan fluorescence of avidin by biotin forms the basis for a rapid, sensitive assay for biotin and avidin in solutions free of fluorescent contaminants. Avidin solutions are assayed by titration with a standard biotin solution until no further decrease in fluorescence at 350 nm, with excitation at 290 nm. is observed. Unknown quantities of biotin in solution may be determined by titration with aliquots of standard avidin until the change in fluorescence observed following each addition undergoes a sharp increase. As little as 20 ng of biotin may be measured in this way.
Several methods for the assay of avidin, biotin, or both are currently available. The increase in the absorptivity of avidin at 233 nm upon the binding of biotin (AeZS3= 2.4 x IO4 M-' cm-’ mol-’ of biotin) (l-3) and the ability of biotin to displace quantitatively 4’-hydroxyazobenzene-2carboxylate from its colored complex with avidin (3,4) provide the bases for two assays for either the protein or biotin. The latter assay is sensitive to about 50 pg of avidin and 1 pug of biotin (3). More sensitive avidin assays have been accomplished using [14C]biotin (5-8) [lower limit = 20 ng of avidin (8)]. Biotin may be assayed chemically, using p -dimethylaminocinnamaldehyde (lower limit = 10 pg) (9,10), or with one of many biological procedures (11). Many organisms are suitable for the biological assays, such as Lactobacillusplantarum (12), Lactobacillus casei (13,14), Neurospora crassa (15), and Ochromonas danica (16) (typical lower limit = 0.01 ng), but these assays may take 3 to 5 days to perform. Green (17) has noted that the binding of biotin to avidin is accompanied by a decrease in the fluorescence intensity of the protein, an observation later confirmed by Chignell and co-workers (18). This suggests that, because of the high affinity of avidin for biotin, these observations may be generalized into an assay for either avidin or biotin. 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. A procedure for the determination of free biotin in amounts as small as 20 ng is described below. 442 Copyright P 1977 by Academr Press. Inc. All rights of reproduction m any form reserved
1SSN ooO3-2697
RAPID ASSAY FOR AVIDIN
AND BIOTIN
443
EXPERIMENTAL
Avidin and d-biotin [mp 229-230°C lit. mp 230-232°C (19)] were purchased from Worthington Biochemical Corp. and from Sigma, respectively. Fluorescence measurements were made with a Hitachi PerkinElmer spectrofluorometer (Model MPF-2A) whose emission monochromator had been calibrated with indole (Aldrich Chemical Company, mp 52-53°C) (20). All measurements were made in the ratio recording mode with the temperature between 20 and 24°C. All solutions were made in 0.15 M sodium phosphate buffer at pH 7, ionic strength = 0.35. 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 pg/ml. Two milliliters of approximately 10 pg/rnl of avidin solution is pipetted into a 4-ml fluorometer cell and titrated with successive 2.0-~1.1aliquots of the standard 20-p.g/ml biotin solution using a syringe microburet (Micrometric Instrument Co., Model SB2). The excitation monochromator is set at 290 nm using a bandwidth of 4 nm, and the fluorescence is monitored after each addition at 350 nm using a bandwidth of 16 nm. Additions are made until no further decrease in fluorescence is observed, indicating the attainment of the equivalence point (Fig. 1). Titration of biotin. Typically, a solution of avidin standardized with biotin as described above is diluted to 250 pg/ml and is used to titrate 2 ml of a solution that contains an unknown amount of biotin in a fluorometer
Biotin/Avidin
[pg/mg]
FIG. 1. Typical titration of avidin with biotin. About 25 fig of avidin in 2.0 ml of 0.15 M sodium phosphate buffer, pH 7, was titrated with 2.0-4 aliquots of a 90 pM (22 &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 (22).
444
LIN AND KIRSCH
0
20 Volume Avidin Solution Added [PI]
30
IO
FIG. 2. Typical titration of biotin with avidin. Biotin, 33 ng in 2.0 ml of 0.15 M sodium phosphate buffer, pH 7, was titrated with 2.0~~1 ahquots of a 250+FLg/ml avidin solution. Fluorescence was measured at 350 nm with excitation at 290 nm.
cell. Relative fluorescence is measured after each addition of 2.03~1 aliquots of the avidin solution from a syringe microburet, 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 point, and the quantity of biotin is determined from the specific activity of the avidin solution (Fig. 2). RESULTS AND DISCUSSION
The fluorescent properties of free avidin and of avidin saturated with biotin are summarized in Table 1. The results of titrating avidin with biotin and of titrating biotin with avidin, as described under Experimental, are illustrated in Figs. 1 and 2. TABLE FLUORESCENT
PROPERTIES
OF FREE
Emission hmaxb(nm) Relative fluorescence at 350 nm (bandwidth = 16 nm)
AVIDIN
1
AND
OF AVIDIN
SATURATED
WITH
BIOTIN”
Free avidin
Avidin saturated with biotin
342
332
100
55 2 0.8
u In 0.15 or 0.25 M Na-phosphate buffers, 20-24°C. with excitation (bandwidth = 4 nm). Avidin concentration = 13 pg/ml. * A shift from 336 to 326 nm is reported by Chignell et al. (18).
at 290 nm
RAPID
ASSAY
FOR
AVIDIN
AND
BIOTIN
445
The quenching of the protein fluorescence by biotin is consistent with the observation that avidin-bound biotin protects all four tryptophan residues of an avidin subunit from oxidation by N-bromosuccinimide (1,2,2 I-23). The avidin specific activities determined by titration of avidin with biotin (11 .O ? 0.2 pg of biotin/mg, seven determinations) and by titration of biotin with avidin (10.8 2 0.2 pg of biotin/mg, four determinations) agree well with that given by the manufacturer (10.6 pg of biotin/mg). 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 l-2 PM. [Biological fluids have high concentrations of tryptophan, e.g., 54 PM in human serum (24) and 69 PM in human urine (25), compared to their concentrations of biotin, about 1 nM in serum and 70 nM in urine (16)]. A few other proteins are known to bind biotin, such as streptavidin (26) and a recently described egg yolk protein (27). 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. ACKNOWLEDGMENT This work Foundation.
was
supported
by
a grant
(BMS
74-17633)
from
the
National
Science
REFERENCES I. Green. N. M. (1962) Biochim. Biophys. Acru 59, 244-246. 2. Green, N. M. (1963) Biochrm. J. 89, 599-609. 3. Green, N. M. (1970)in Methods in Enzymology (McCormick. D. B.. and Wright. L. D.. eds.). Vol. 18A. pp. 418-424. Academic Press, New York. 4. Green, N. M. (1965) Biochem. J. 94, 23C-24C. 5. Wei, R. D., and Wright. L. D. (1964) Proc. Sot. Exp. Biol. Med. 117, 17-20. 6. Wei. R. D. (1970) in Methods in Enzymology (McCormick, D. B., and Wright, L. D., eds.). Vol. 18A. pp. 424-427, Academic Press, New York. 7. Korenman. S. G., and O’Malley, B. W. (1967) Bio&‘m. Biophys. Ac-ru 140, 174-176. 8. Korenman. S. G., and O’Malley. B. W. (1970)in Methods in Enzymology (McCormick. D. B.. and Wright. L. D.. eds.). Vol. 18A, pp. 427-430, Academic Press. New York. 9. McCormick. D. B., and Roth, J. A. (1970) Anal. Biochem. 34, 226-236. IO. McCormick, D. B., and Roth, J. A. (1970) in Methods in Enzymology (McCormick, D. B., and Wright, L. D.. eds.). Vol. 18A, pp. 383-385, Academic Press, New York. 11. Gyiirgy. P., and Langer. B. W.. Jr. (1968) in The Vitamins (Sebrell, W. H., Jr.. and Harris, R. S.. eds.). 2nd ed., Vol. 2. pp. 2X0-284, Academic Press. New York.
446
LIN
AND
KIRSCH
12. Wright, L. D., and Skeggs. H. R. (1944) Proc. Sot. Exp. Biol. Med. 56, 95-98. 13. Shull, G. M.. Hutchings. B. L., and Peterson, W. H. (1942) .I. Bio/. Chem. 142, 913-920. 14. Shull. G. M.. and Peterson, W. H. (1943) J. Biol. Chem. 151, 201-202. 15. Hodson. A. Z. (1945) J. Biol. Chem. 157, 383-385. 16. Baker, H.. Frank, O., Matovitch, V. B.. Pasher, I., Aaronson. S., Hutner, S. H., and Sobotka, H. (1962) Anal. Biochem. 3, 31-39. 17. Green, N. M. (1964) Biochem. .I. 90, 564-568. 18. Chignell, C. F.. Starkweather, D. K.. and Sinha, B. K. (1975) J. Biol. Chem. 250, 5622-5630.
19. du Vigneaud. V., Hofmann, K., Melville, D. B., and Rachele, J. R. (1941) J. Bio/. Chem. 140, 763-766. 20. Berlman. I. B. (1%5) Handbook of Fluorescence Spectra of Aromatic Molecules, p. 101, Academic Press,New York. 21. Green, N. M., and Toms, E. J. (1972) Biochem. J. 130, 707-711. 22. Green, N. M. (1964) Biochem. J. 92, l6C-l7C. 23. DeLange, R. J., and Huang, T. S. (1971) J. Biol. Chem. 246, 698-709. 24. Hier. S. W.. and Bergeim. 0. (1946) J. Biol. Chem. 163, 129-135. 25. Woodson, H. W., Hier. S. W., Solomon, J. D., and Bergeim, 0. (1948) J. Biol. Chem. 172, 613-618. 26. Chaiet, L., and Wolf, F. J. (1964) Arch. Biochem. Biophys. 106, l-5. 27. White. H. B., Dennison, B. A.. Della Fera. M. A., Whitney, C. J., McGuire, J. C.. Meslar. H. W.. and Sammelwitz, P. H. (1976) Biochem. J. 157, 395-400.