234
GENERALMETHODOLOGY
[25]
[25] F l u o r o m e t r i c A s s a y for A v i d i n - B i o t i n I n t e r a c t i o n
By DONALD M. MOCK and PAUL HOROWITZ Absolute quantitation of biotin and avidin can be important in many applications of avidin-biotin technology and in studies of biotin nutriture. Many assays for biotin in physiological fluids such as blood and urine are based on the interaction of biotin with avidin and require standardization of the avidin to be used in the assay. Biotin can be quantitated by weight, and then avidin can be quantitated by measuring changes in the optical absorbance spectrum that occur when biotin displaces 4'-hydroxyazobenzene-2-carboxylic acid (HABA) from avidin. ~ An alternate approach is quantitation of avidin by absorbance at 280 nm or by changes in the absorbance spectrum that occur when HABA binds to avidin; biotin can then be quantitated by the optical absorbance changes that occur with displacement of HABA from avidin by biotin. The interaction of avidin and biotin can also be quantitated by measuring the fluorescence changes that occur when the fluorescent probe 2anilinonaphthalene-6-sulfonic acid (2,6-ANS) is displaced by biotin from the biotin-binding site on avidin. 2 The probe probably binds to a hydrophobic region at or near the biotin-binding site on avidin. The transition from an aqueous to a hydrophobic environment is associated with a greater than 6-fold increase in the fluorescence intensity, and the wavelength of maximum emission shifts from 463 to 422 nm. When biotin is added, it stoichiometrically displaces the 2,6-ANS (mole ratio 1 : 1) and reverses the enhancement of fluorescence intensity and the shift in wavelength of maximum emission. This fluorometric method is approximately an order of magnitude more sensitive than the HABA method and is less subject to interference from naturally occurring chromophores; the method is also relatively insensitive to naturally occurring fluorophores. Although this method can be used to standardize either avidin or biotin, for simplicity the standardization of avidin is described in this chapter. The differences when used to standardize biotin are briefly discussed at the end of the chapter. 1 N. M. Green, Biochem. J. 94, 23c (1965). 2 D. M. Mock, G. Lankford, D. DuBois, N. Criscimagna, and P. Horowitz, Anal. Biochem. 151, 178 (1985).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[25]
FLUOROMETRICASSAY
235
Assay Method
Materials. 2,6-ANS is available commercially from Molecular Probes (Junction City, OR). Avidin-D is affinity purified and available commercially from Vector Laboratories (Burlingame, CA). Biotin (d-biotin) is from Sigma Chemical Co. (St. Louis, MO). Because the fluorescence of the probe and perhaps even the interaction of the probe and avidin may be sensitive to pH, quantitation should be conducted in an appropriate buffer. 3 Equipment. We have successfully used two spectrophotofluorometers for this standardization: (1) Farand Mark V spectrophotofluorometers and (2) Shimadzu RF-540 recording spectrophotofluorometers. The excitation wavelength is 328 nm, the emission wavelength 408 nm. A slit width of 5 nm for both excitation and emission beams is suitable. The wavelength of maximum intensity depends on the solvent system in which the probe is dissolved. Because the equilibrium dissociation constant is about 200/zM, neither the bound nor the free probe concentrations are negligible with respect to the other at the concentrations typically used. Thus, the wavelength of maximum intensity of the probeavidin complex has been determined by extrapolation to infinite avidin concentration (i.e., all probe bound). The wavelengths are chosen to maximize the difference in fluorescence intensity between bound and free 2,6ANS based on published maxima for excitation and emission while avoiding scattered light from the excitation beam. In the buffer system used, the true maximum wavelength of emission of free 2,6-ANS is 463 nm and that of bound 2,6-ANS 422 nm. Sensitivity is still satisfactory when monitoring emission at 408 nm; thus, within limits, the choice of excitation and emission wavelengths is not critical. Titration of 2,6-ANS-Avidin Complex by Biotin Initially, avidin and 2,6-ANS are added to a quartz cuvette, mixed, and placed in the fluorometer. The maximum fluorescence enhancement is reached in the few seconds required for mixing and insertion of the cuvette; no extra incubation time is required at this point or during the titration. Biotin is then added in increments from a concentrated stock solution, the components are mixed, and the fluorescence intensity is measured after each addition. A typical starting volume is 2.00 ml, and a typical titration would be fifteen 10-/A additions of a 0.12 mM biotin s We chose 0.2 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES Ultrol, Calbiochem, La Jolla, CA), pH 7.0, in 2 M NaC1 because this buffer gives a satisfactory avidin-biotin interaction in a biotin assay (see D. M. Mock, this volume [24]).
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GENERALMETHODOLOGY
[25]
solution in deionized, distilled water. Biotin does not dissolve easily in water at concentrations greater than 2 mg/ml ( - 8 mM). It may be necessary to make the solution slightly basic with NaHCO3 or NaOH to facilitate dissolving the biotin and then correct back to pH 7.0 with HC1. The choice of concentration of avidin (or amount of dilution of the unknown avidin source) depends on at least two factors: (1) the sensitivity of the fluorometer and (2) the optical path length of the cuvette. Most fluorescence cuvettes have an optical path length of 1 cm. For experiments requiring high concentrations of 2,6-ANS and thus producing high inner filter effects, we have successfully used a flow cell (Shimadzu RF-540 flow cell, 2040382504) with a path length of 2 mm. When initially setting up the method, it is worthwhile to vary the concentrations of avidin and of 2,6-ANS in the cuvette to achieve the following result when a saturating amount of biotin is added: The decrease in fluorescence intensity should be great enough for accurate quantitation (e.g., -+2% reproducibility) on the available fluorometer. A "saturating" biotin concentration is at least 4-fold greater than the avidin concentration. Typical avidin concentrations are 1-200/zM. Typical 2,6>. I--
30 Q
Z w z w 20 0 z w 0 o9 I,Ll IO
~
-J
A
A
•
•
•
•
0
er CONCENTRATION OF BIOTIN (pM) FTG. I. Titrations of the complex between avidin and 2,6-ANS with biotin at several concentrations of 2,6-ANS. The corrected fluorescence intensity is plotted as a function of the concentrationof biotin. The 2,6-ANS concentrationsshown are 94.1 IzM (top curve),
47.0/zM (middlecurve), and 23.5/zM (bottom curve). The avidinconcentrationis 1.74/zM in all three titrations.
[25]
FLUOROMETRIC ASSAY
237
ANS concentrations are roughly equimolar; the point of equivalence (hence, the accuracy of the method) is not dependent on the 2,6-ANS concentration over a fairly wide range (Fig. 1), because the association constant for biotin and avidin is very high ( K a = l015 M-I). For the same reason, the accuracy of this method is not affected by the concentration of avidin in the cuvette (Fig. 2) unless the avidin concentration is too small for accurate quantitation of the fluorescence change or so great that the accompanying high concentrations of 2,6-ANS cause an inner filter effect so large that fluorescence increases cannot be accurately measured and corrected. The point of equivalence is defined as the point at which all binding sites on avidin are occupied by biotin with no excess free biotin. Because of the high binding affinity, the effects due to equilibrium are negligible at the concentrations used for this method. However, nonlinearity of the >I--
Z LU IZ
20
16
I11
Z
I11 (D
12
LU
0 ,_1 LI. Ill w
--
I-..I
I.U 0
|
l
I
I
I
I
0.2
0.4
0.6
0.8
1.0
1.2
14
1.
|
i
1.6
1.8
iB'OT'NVIAV'D'Ni FIG. 2. Titration of the complex between avidin and 2,6-ANS with biotin at several avidin concentrations. The corrected fluorescence intensity is plotted as a function of the ratio of the biotin concentration to the monomeric avidin concentration. The avidin concentrations, determined independently by optical absorbance at 280 nm, are 1, 4, 6, and 8 / z M from bottommost curve to topmost. The 2,6-ANS concentration is fixed at 50 p,M. The point of equivalence should be 1.0, but this particular batch of avidin consistently demonstrates a value of about 0.75.
238
GENERAL METHODOLOGY
[25]
titration curve near the equivalence point is sometimes seen. Whether this represents heterogeneity in the avidin preparation or cooperativity in the avidin tetramer (i.e., Ka of one site depends on the number of sites already occupied in that avidin molecule) is not clear. The point of equivalence can be determined graphically or by linear regression as the point of intersection of the two linear segments of the titration curve; if nonlinearity is seen near the equivalence point, these points should be excluded from the linear regression. The unknown avidin concentration at the point of equivalence [A]e is related to the biotin concentration at the point of equivalence [B]e by the following equation: [A]e = [B]e/4 Keep in mind that the titration may have added significantly to the volume in the cuvette. An alternate equation for the original avidin concentration in the cuvette [A]o (in/~mol/liter) is the following: [A]o = biotin/4Vi where biotin is the total micromoles of biotin added at the equivalence point and is equal to the volume of biotin stock added (/zl) times the concentration of biotin stock (in/zmol//xl), and VI is the initial volume in the cuvette (in liters; e.g., 2.00 x 10-3 liter). The factor of 4 reflects the assumption that all four sites of tetrameric avidin are available for biotin binding. Using absorbance at 280 nm to standardize avidin independently, 2 we have occasionally found samples of affinity-purified avidin that had three rather than four biotin-binding sites available (Fig. 2). This unusual stoichiometry was confirmed by HABA titration. 2 This finding emphasizes the caveat that multimeric binding proteins need only a single functional binding site to be purified by affinity methods. Biotin contamination could result in essentially irreversible occupation of approximately 25% of the sites in a given lot of avidin. Inner filter effects become important when the number of excitation photons reaching the fluorophores in the region of the cuvette monitored for emission is significantly reduced because of the absorption by fluorophores encountered earlier in the monitored region; at this point, the fluorescence intensity is no longer linear with concentration of fluorophore. Since changes in fluorescence intensity are linear with added biotin in the range of 2,6-ANS concentrations used, the same equivalence point will be determined whether or not the fluorescence intensity is corrected for the inner filter effect. For experiments measuring the binding characteristics of the probe, however, a correction factor according to
[25]
FLUOROMETRIC ASSAY
239
the equation of McClure and Edelman 4 is necessary: Icorr ---- lob s ×
2.303Aex/(l
-
l 0 -aex)
where Icorr is the corrected fluorescence intensity, lobs the observed fluorescence intensity, and Aex the absorbance of the probe at the excitation wavelength. Aex is calculated as the product of the extinction coefficient at the excitation wavelength times optical path length times concentration of probe. This fluorescence method will give erroneously low values for avidin if significant amounts of biotin or biotin analogs are present in the avidin sample, provided that the biotin analogs have an intact heterocyclic ring structure and thus bind as tightly to avidin as biotin. Biocytin and biotin methyl ester are examples of such biotin analogs. The effect of biotin analogs with altered ring structures (e.g., dethiobiotin, biotin sulfoxide, diaminobiotin) on this method has not been studied in detail.
Titration of Biotin by Avidin-2,6-ANS Complex To use the fluorometric method to measure biotin, the source of avidin must first be standardized. With a pure preparation, absorbance at 280 nm can be measured, or the avidin can be quantitated by weight. Alternatively, a primary biotin standard can be used to standardize the avidin, and the avidin, in turn, used to standardize samples of unknown biotin concentration. The titrations to determine the equivalence point are conducted as discussed above if the concentration of biotin in the unknown sample is sufficiently large. If not, a concentrated solution containing avidin and 2,6-ANS can be used to titrate the biotin solution. In this reverse titration, the fluorescence should first increase linearly at a modest slope with sequential additions of avidin-2,6-ANS solution because biotin will immediately displace all the probe into the aqueous phase; at the equivalence point, the slope will increase dramatically because no free biotin remains to displace the probe. Biotin concentration is calculated as follows: [B]e = [Ale x 4
This reverse titration (i.e., biotin titrated by avidin) also works if 2,6ANS is added to the cuvette containing the unknown concentration of biotin rather than along with the avidin. Because the amount of free probe changes little with the avidin additions (except for dilution) until the point 4 W. O. McClure and G. M. Edelman, Anal. Biochem. 6, 559 (1967).
240
GENERAL METHODOLOGY
[25]
of equivalence is reached, the fluorescence intensity increases only after reaching the point of equivalence. Thus, the method of reverse titration may offer a minor advantage in precision compared to titrating with avidin-2,6-ANS solution. Neither fluorescence enhancement nor reversal is seen with the interaction of 2,6-ANS, streptavidin, and biotin.
GENERALMETHODOLOGY
[25]
[25] F l u o r o m e t r i c A s s a y for A v i d i n - B i o t i n I n t e r a c t i o n
By DONALD M. MOCK and PAUL HOROWITZ Absolute quantitation of biotin and avidin can be important in many applications of avidin-biotin technology and in studies of biotin nutriture. Many assays for biotin in physiological fluids such as blood and urine are based on the interaction of biotin with avidin and require standardization of the avidin to be used in the assay. Biotin can be quantitated by weight, and then avidin can be quantitated by measuring changes in the optical absorbance spectrum that occur when biotin displaces 4'-hydroxyazobenzene-2-carboxylic acid (HABA) from avidin. ~ An alternate approach is quantitation of avidin by absorbance at 280 nm or by changes in the absorbance spectrum that occur when HABA binds to avidin; biotin can then be quantitated by the optical absorbance changes that occur with displacement of HABA from avidin by biotin. The interaction of avidin and biotin can also be quantitated by measuring the fluorescence changes that occur when the fluorescent probe 2anilinonaphthalene-6-sulfonic acid (2,6-ANS) is displaced by biotin from the biotin-binding site on avidin. 2 The probe probably binds to a hydrophobic region at or near the biotin-binding site on avidin. The transition from an aqueous to a hydrophobic environment is associated with a greater than 6-fold increase in the fluorescence intensity, and the wavelength of maximum emission shifts from 463 to 422 nm. When biotin is added, it stoichiometrically displaces the 2,6-ANS (mole ratio 1 : 1) and reverses the enhancement of fluorescence intensity and the shift in wavelength of maximum emission. This fluorometric method is approximately an order of magnitude more sensitive than the HABA method and is less subject to interference from naturally occurring chromophores; the method is also relatively insensitive to naturally occurring fluorophores. Although this method can be used to standardize either avidin or biotin, for simplicity the standardization of avidin is described in this chapter. The differences when used to standardize biotin are briefly discussed at the end of the chapter. 1 N. M. Green, Biochem. J. 94, 23c (1965). 2 D. M. Mock, G. Lankford, D. DuBois, N. Criscimagna, and P. Horowitz, Anal. Biochem. 151, 178 (1985).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[25]
FLUOROMETRICASSAY
235
Assay Method
Materials. 2,6-ANS is available commercially from Molecular Probes (Junction City, OR). Avidin-D is affinity purified and available commercially from Vector Laboratories (Burlingame, CA). Biotin (d-biotin) is from Sigma Chemical Co. (St. Louis, MO). Because the fluorescence of the probe and perhaps even the interaction of the probe and avidin may be sensitive to pH, quantitation should be conducted in an appropriate buffer. 3 Equipment. We have successfully used two spectrophotofluorometers for this standardization: (1) Farand Mark V spectrophotofluorometers and (2) Shimadzu RF-540 recording spectrophotofluorometers. The excitation wavelength is 328 nm, the emission wavelength 408 nm. A slit width of 5 nm for both excitation and emission beams is suitable. The wavelength of maximum intensity depends on the solvent system in which the probe is dissolved. Because the equilibrium dissociation constant is about 200/zM, neither the bound nor the free probe concentrations are negligible with respect to the other at the concentrations typically used. Thus, the wavelength of maximum intensity of the probeavidin complex has been determined by extrapolation to infinite avidin concentration (i.e., all probe bound). The wavelengths are chosen to maximize the difference in fluorescence intensity between bound and free 2,6ANS based on published maxima for excitation and emission while avoiding scattered light from the excitation beam. In the buffer system used, the true maximum wavelength of emission of free 2,6-ANS is 463 nm and that of bound 2,6-ANS 422 nm. Sensitivity is still satisfactory when monitoring emission at 408 nm; thus, within limits, the choice of excitation and emission wavelengths is not critical. Titration of 2,6-ANS-Avidin Complex by Biotin Initially, avidin and 2,6-ANS are added to a quartz cuvette, mixed, and placed in the fluorometer. The maximum fluorescence enhancement is reached in the few seconds required for mixing and insertion of the cuvette; no extra incubation time is required at this point or during the titration. Biotin is then added in increments from a concentrated stock solution, the components are mixed, and the fluorescence intensity is measured after each addition. A typical starting volume is 2.00 ml, and a typical titration would be fifteen 10-/A additions of a 0.12 mM biotin s We chose 0.2 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES Ultrol, Calbiochem, La Jolla, CA), pH 7.0, in 2 M NaC1 because this buffer gives a satisfactory avidin-biotin interaction in a biotin assay (see D. M. Mock, this volume [24]).
236
GENERALMETHODOLOGY
[25]
solution in deionized, distilled water. Biotin does not dissolve easily in water at concentrations greater than 2 mg/ml ( - 8 mM). It may be necessary to make the solution slightly basic with NaHCO3 or NaOH to facilitate dissolving the biotin and then correct back to pH 7.0 with HC1. The choice of concentration of avidin (or amount of dilution of the unknown avidin source) depends on at least two factors: (1) the sensitivity of the fluorometer and (2) the optical path length of the cuvette. Most fluorescence cuvettes have an optical path length of 1 cm. For experiments requiring high concentrations of 2,6-ANS and thus producing high inner filter effects, we have successfully used a flow cell (Shimadzu RF-540 flow cell, 2040382504) with a path length of 2 mm. When initially setting up the method, it is worthwhile to vary the concentrations of avidin and of 2,6-ANS in the cuvette to achieve the following result when a saturating amount of biotin is added: The decrease in fluorescence intensity should be great enough for accurate quantitation (e.g., -+2% reproducibility) on the available fluorometer. A "saturating" biotin concentration is at least 4-fold greater than the avidin concentration. Typical avidin concentrations are 1-200/zM. Typical 2,6>. I--
30 Q
Z w z w 20 0 z w 0 o9 I,Ll IO
~
-J
A
A
•
•
•
•
0
er CONCENTRATION OF BIOTIN (pM) FTG. I. Titrations of the complex between avidin and 2,6-ANS with biotin at several concentrations of 2,6-ANS. The corrected fluorescence intensity is plotted as a function of the concentrationof biotin. The 2,6-ANS concentrationsshown are 94.1 IzM (top curve),
47.0/zM (middlecurve), and 23.5/zM (bottom curve). The avidinconcentrationis 1.74/zM in all three titrations.
[25]
FLUOROMETRIC ASSAY
237
ANS concentrations are roughly equimolar; the point of equivalence (hence, the accuracy of the method) is not dependent on the 2,6-ANS concentration over a fairly wide range (Fig. 1), because the association constant for biotin and avidin is very high ( K a = l015 M-I). For the same reason, the accuracy of this method is not affected by the concentration of avidin in the cuvette (Fig. 2) unless the avidin concentration is too small for accurate quantitation of the fluorescence change or so great that the accompanying high concentrations of 2,6-ANS cause an inner filter effect so large that fluorescence increases cannot be accurately measured and corrected. The point of equivalence is defined as the point at which all binding sites on avidin are occupied by biotin with no excess free biotin. Because of the high binding affinity, the effects due to equilibrium are negligible at the concentrations used for this method. However, nonlinearity of the >I--
Z LU IZ
20
16
I11
Z
I11 (D
12
LU
0 ,_1 LI. Ill w
--
I-..I
I.U 0
|
l
I
I
I
I
0.2
0.4
0.6
0.8
1.0
1.2
14
1.
|
i
1.6
1.8
iB'OT'NVIAV'D'Ni FIG. 2. Titration of the complex between avidin and 2,6-ANS with biotin at several avidin concentrations. The corrected fluorescence intensity is plotted as a function of the ratio of the biotin concentration to the monomeric avidin concentration. The avidin concentrations, determined independently by optical absorbance at 280 nm, are 1, 4, 6, and 8 / z M from bottommost curve to topmost. The 2,6-ANS concentration is fixed at 50 p,M. The point of equivalence should be 1.0, but this particular batch of avidin consistently demonstrates a value of about 0.75.
238
GENERAL METHODOLOGY
[25]
titration curve near the equivalence point is sometimes seen. Whether this represents heterogeneity in the avidin preparation or cooperativity in the avidin tetramer (i.e., Ka of one site depends on the number of sites already occupied in that avidin molecule) is not clear. The point of equivalence can be determined graphically or by linear regression as the point of intersection of the two linear segments of the titration curve; if nonlinearity is seen near the equivalence point, these points should be excluded from the linear regression. The unknown avidin concentration at the point of equivalence [A]e is related to the biotin concentration at the point of equivalence [B]e by the following equation: [A]e = [B]e/4 Keep in mind that the titration may have added significantly to the volume in the cuvette. An alternate equation for the original avidin concentration in the cuvette [A]o (in/~mol/liter) is the following: [A]o = biotin/4Vi where biotin is the total micromoles of biotin added at the equivalence point and is equal to the volume of biotin stock added (/zl) times the concentration of biotin stock (in/zmol//xl), and VI is the initial volume in the cuvette (in liters; e.g., 2.00 x 10-3 liter). The factor of 4 reflects the assumption that all four sites of tetrameric avidin are available for biotin binding. Using absorbance at 280 nm to standardize avidin independently, 2 we have occasionally found samples of affinity-purified avidin that had three rather than four biotin-binding sites available (Fig. 2). This unusual stoichiometry was confirmed by HABA titration. 2 This finding emphasizes the caveat that multimeric binding proteins need only a single functional binding site to be purified by affinity methods. Biotin contamination could result in essentially irreversible occupation of approximately 25% of the sites in a given lot of avidin. Inner filter effects become important when the number of excitation photons reaching the fluorophores in the region of the cuvette monitored for emission is significantly reduced because of the absorption by fluorophores encountered earlier in the monitored region; at this point, the fluorescence intensity is no longer linear with concentration of fluorophore. Since changes in fluorescence intensity are linear with added biotin in the range of 2,6-ANS concentrations used, the same equivalence point will be determined whether or not the fluorescence intensity is corrected for the inner filter effect. For experiments measuring the binding characteristics of the probe, however, a correction factor according to
[25]
FLUOROMETRIC ASSAY
239
the equation of McClure and Edelman 4 is necessary: Icorr ---- lob s ×
2.303Aex/(l
-
l 0 -aex)
where Icorr is the corrected fluorescence intensity, lobs the observed fluorescence intensity, and Aex the absorbance of the probe at the excitation wavelength. Aex is calculated as the product of the extinction coefficient at the excitation wavelength times optical path length times concentration of probe. This fluorescence method will give erroneously low values for avidin if significant amounts of biotin or biotin analogs are present in the avidin sample, provided that the biotin analogs have an intact heterocyclic ring structure and thus bind as tightly to avidin as biotin. Biocytin and biotin methyl ester are examples of such biotin analogs. The effect of biotin analogs with altered ring structures (e.g., dethiobiotin, biotin sulfoxide, diaminobiotin) on this method has not been studied in detail.
Titration of Biotin by Avidin-2,6-ANS Complex To use the fluorometric method to measure biotin, the source of avidin must first be standardized. With a pure preparation, absorbance at 280 nm can be measured, or the avidin can be quantitated by weight. Alternatively, a primary biotin standard can be used to standardize the avidin, and the avidin, in turn, used to standardize samples of unknown biotin concentration. The titrations to determine the equivalence point are conducted as discussed above if the concentration of biotin in the unknown sample is sufficiently large. If not, a concentrated solution containing avidin and 2,6-ANS can be used to titrate the biotin solution. In this reverse titration, the fluorescence should first increase linearly at a modest slope with sequential additions of avidin-2,6-ANS solution because biotin will immediately displace all the probe into the aqueous phase; at the equivalence point, the slope will increase dramatically because no free biotin remains to displace the probe. Biotin concentration is calculated as follows: [B]e = [Ale x 4
This reverse titration (i.e., biotin titrated by avidin) also works if 2,6ANS is added to the cuvette containing the unknown concentration of biotin rather than along with the avidin. Because the amount of free probe changes little with the avidin additions (except for dilution) until the point 4 W. O. McClure and G. M. Edelman, Anal. Biochem. 6, 559 (1967).
240
GENERAL METHODOLOGY
[25]
of equivalence is reached, the fluorescence intensity increases only after reaching the point of equivalence. Thus, the method of reverse titration may offer a minor advantage in precision compared to titrating with avidin-2,6-ANS solution. Neither fluorescence enhancement nor reversal is seen with the interaction of 2,6-ANS, streptavidin, and biotin.
