Homogeneous competitive assay of ligand affinities based on quenching fluorescence of tyrosine/tryptophan residues in a protein via Frster-resonance-energy-transfer

Article abstract of DOI:10.1016/j.saa.2010.08.021

A new homogeneous competitive assay of ligand affinities was proposed based on quenching the fluorescence of tryptophan/tyrosine residues in a protein via Frster-resonance-energy-transfer using a fluorescent reference ligand as the acceptor. Under excitation around 280 nm, the fluorescence of a protein or a bound acceptor was monitored upon competitive binding against a nonfluorescent candidate ligand. Chemometrics for deriving the binding ratio of the acceptor with either fluorescence signal was discussed; the dissociation constant (K _d) of a nonfluorescent candidate ligand was calculated from its concentration to displace 50% binding of the acceptor. N-biotinyl-N′-(1- naphthyl)-ethylenediamine (BNEDA) and N-biotinyl-N′-dansyl-ethylenediamine (BDEDA) were used as the reference ligands and acceptors to streptavidin to test this new homogeneous competitive assay. Upon binding of an acceptor to streptavidin, there were the quench of streptavidin fluorescence at 340 nm and the characteristic fluorescence at 430 nm for BNEDA or at 525 nm for BDEDA. K_d of BNEDA and BDEDA was obtained via competitive binding against biotin. By quantifying BNEDA fluorescence, K_d of each tested nonfluorescent biotin derivative was consistent with that by quantifying streptavidin fluorescence using BNEDA or BDEDA as the acceptor. The overall coefficients of variation were about 10%. Therefore, this homogeneous competitive assay was effective and promising to high-throughput-screening.

Full text of DOI:10.1016/j.saa.2010.08.021

Spectrochimica Acta Part A 77 (2010) 869–876
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Homogeneous competitive assay of ligand affinities based on quenching
fluorescence of tyrosine/tryptophan residues in a protein via
Fo˝ rster-resonance-energy-transfer
Yanling Xiea,1 , Xiaolan Yanga,1 , Jun Pua , Yunsheng Zhaoa , Ying Zhanga , Guoming Xieb , Jun Zhengb ,
Huidong Yuana, Fei Liaoa,∗
a Unit for Biotransformation and Protein Biotechnology, Chongqing Key Laboratory of Biochemical & Molecular Pharmacology, Chongqing Medical University, Chongqing 400016,
China
b Key Laboratory of Laboratory Medical Diagnostics of the Ministry of Education, Chongqing Medical University, Chongqing 400016, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 May 2010
Accepted 5 August 2010
A new homogeneous competitive assay of ligand affinities was proposed based on quenching the flu-
orescence of tryptophan/tyrosine residues in a protein via Fo˝ rster-resonance-energy-transfer using a
fluorescent reference ligand as the acceptor. Under excitation around 280 nm, the fluorescence of a pro-
tein or a bound acceptor was monitored upon competitive binding against a nonfluorescent candidate
ligand. Chemometrics for deriving the binding ratio of the acceptor with either fluorescence signal was
discussed; the dissociation constant (Kd) of a nonfluorescent candidate ligand was calculated from its con-
centration to displace 50% binding of the acceptor. N-biotinyl-Nꢀ-(1-naphthyl)-ethylenediamine (BNEDA)
and N-biotinyl-Nꢀ-dansyl-ethylenediamine (BDEDA) were used as the reference ligands and acceptors to
streptavidin to test this new homogeneous competitive assay. Upon binding of an acceptor to strepta-
vidin, there were the quench of streptavidin fluorescence at 340 nm and the characteristic fluorescence at
430 nm for BNEDA or at 525 nm for BDEDA. Kd of BNEDA and BDEDA was obtained via competitive bind-
ing against biotin. By quantifying BNEDA fluorescence, Kd of each tested nonfluorescent biotin derivative
was consistent with that by quantifying streptavidin fluorescence using BNEDA or BDEDA as the acceptor.
The overall coefficients of variation were about 10%. Therefore, this homogeneous competitive assay was
effective and promising to high-throughput-screening.
Keywords:
Biotin derivatives
Binding ratio
Fo˝ rster-resonance-energy-transfer
Dissociation constant
Streptavidin
Tryptophan residues
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
ligand against a reference ligand labeled with radio-active iso-
topes or fluorophores to facilitate selective quantification of the
Ligands of proteins play important roles in biomedicine and
their affinities to proteins, i.e., dissociation constants of the com-
plexes between proteins and ligands, primarily determine their
biomedical significance. To determine the affinity of a ligand to
a protein, common methods employ competitive binding of the
complexes in reaction mixtures. In general, competitive assays of
ligand affinities include the heterogeneous method that separates
the complexes from reaction solutions before quantification, and
the homogenous method that quantifies the complexes directly
in reaction solutions without any separation process. In practice,
homogenous competitive assays of ligand affinities are favored for
the efficiency, cost and suitability for high-throughput-screening
(HTS) of ligands in a library, and they are realized principally with
fluorescent reference ligands in either of the following two ways
[1–7]. The first way measures changes of fluorescence polarization
[1,7–13], the second way measures changes of fluorescence due
to Fo˝ rster-resonance-energy-transfer (FRET) [2–6,14–18], upon the
binding of a fluorescent reference ligand to a protein. Usually, the
FRET-based homogenous competitive assay is preferable for its
sensitivity, cost and no dependence on specialized instrumental
items.
Abbreviations: SAV, streptavidin; BBZA, N-biotinyl-benzylamine; BME, methyl
biotinyl ester; BDEDA, N-biotinyl-Nꢀ-dansyl-ethylenediamine; BETA, N-biotinyl-
ethanolamine; BDETA, N-biotinyl-diethanolamine; NHS, N-hydroxylsuccinamide
(NHS); NHS-Biotin, N-hydroxylsuccinamide biotinyl ester; BCHA, N-biotinyl-
cyclohexylamine; BNEDA, N-biotinyl-N-(1-naphthyl)-ethylenediamine; BMPL, N-
biotinyl-morpholine; BR, the binding ratio of the reference ligand; EC50, the
concentration of a candidate ligand for 50% binding of a reference ligand; HTS,
high-throughput-screening.
∗
Corresponding author at: Chongqing Key Laboratory of Biochemical & Molecular
Pharmacology, College of Pharmaceutical Science, Chongqing Medical University,
Chongqing 400016, China. Tel.: +86 23 68485161; fax: +86 23 68485161.
E-mail address: liaofeish@cqmu.edu.cn (F. Liao).
Nowadays, in classical FRET-based homogenous competitive
assay of ligand affinities, amino acid residue(s) on a protein is/are
1
Yanling Xie and Xiaolan Yang contributed equally to this work.
1386-1425/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2010.08.021

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Y. Xie et al. / Spectrochimica Acta Part A 77 (2010) 869–876
Scheme 1. Principle of this new homogeneous competitive assay of ligand affinity.
labeled with a suitable fluorophore to act as acceptor(s) (donor(s))
while a suitable fluorescent reference ligand acts as the donor
(acceptor); in the complexes, the distance between the acceptor(s)
and the donor(s) is so short that FRET is greatly enhanced to quench
fluorescence of the donor(s) and to produce the characteristic fluo-
rescence of the acceptor(s) [2–6,14–18]. In practice, however, this
classical homogenous competitive assay of ligand affinities for HTS
tolerates the cost and time for labeling proteins, potential alter-
ation of protein functions by labeling and too many false positive
results.
Surely, a FRET-based homogenous competitive assay of ligand
affinities is more desirable if the labeling of a protein with a flu-
orophore is not required so that there will be neither cost/time
for the labeling nor the potential alteration on protein functions.
In fact, proteins of reasonable sizes contain tyrosine/tryptophan
residues that account for the intrinsic fluorescence of cofactorless
proteins [6]. In theory, tyrosine/tryptophan residues in a protein
can act as intrinsic donors and FRET is expected as long as a suit-
able fluorescent reference ligand as the FRET acceptor is bound
by the protein. Thus, a new FRET-based homogeneous competi-
tive assay of ligand affinities can be developed by measuring the
characteristic fluorescence emission of the bound FRET acceptor or
the quenched fluorescence of the protein (Scheme 1). In this new
homogeneous competitive assay, there is/are neither cost/time for
labeling proteins nor problems associated with labeling, and con-
ventional fluorospectrometers can be used to measure steady-state
fluorescence with ease.
The quench of fluorescence of tryptophan residues in proteins
via FRET is in common use to study protein conformation [19–26],
but is only occasionally used to quantify ligands [27] and esti-
mate affinities of fluorescent ligands via titration rather than by
competitive binding [28]. As far as we knew, homogeneous com-
petitive assay of affinities of nonfluorescent ligands to proteins
using suitable fluorescent reference ligands as the FRET accep-
tors and tyrosine/tryptophan residues as intrinsic donors has not
been reported. During competitive binding, the concentration of
a candidate ligand to displace 50% binding of a reference lig-
and (EC50) is usually estimated to derive its dissociation constant
[29,30]. The binding of biotin derivatives to streptavidin (SAV)
is widely used in bioaffinity recognition, and convenient meth-
ods to estimate affinities of biotin derivatives are needed [31–33].
The guideline to design fluorescent ligands as FRET acceptors to
tyrosine/tryptophan residues in proteins as the intrinsic donors is
proposed [34,35], and it is easier to design fluorescent biotin deriva-

Y. Xie et al. / Spectrochimica Acta Part A 77 (2010) 869–876
871
tives as the FRET acceptor to SAV [35–38]. In this report, therefore,
we discussed chemometrics to derive EC50 of nonfluorescent can-
didate ligands with this homogeneous competitive assay, and two
fluorescent biotin derivatives were used as reference ligands and
acceptors to test this homogeneous competitive assay by cross-
validation.
2. Materials and methods
2.1. Chemicals
d-Biotin was from Biosci Basic Inc. Streptavidin (SAV) was
from Promega (Z7041). N-Hydroxylsuccinamide (NHS), dansyl
chloride, dicyclohexylcarbodiimide (DCC), and N-(1-naphthyl)-
ethylenediamine (NEDA) were from Alfa Aesar. Morpholine,
cyclohexylamine, benzylamine, ethanolamine, diethanolamine
and other chemicals were domestic analytical products. Water was
restilled before use.
2.2. Instruments
A Shimadzu RF5301PC fluorospectrometer was used to record
the spectra with the excitation slit at 5 nm and the emission slit at
10 nm. To estimate ligand affinities, a Shimadzu RF540 fluorospec-
trometer was routinely used to measure the fluorescence with both
the excitation slit and the emission slit at 10 nm, unless otherwise
stated. API QSTAR LC-Q-TOF was used for electro-spray-ionization
high-resolution-mass-spectrometry (ESI-HRMS) to determine for-
mula composition, and DRX 500 M Hz NMR spectrometer was used
to record NMR data (Kunming Institute of Botany, Chinese Academy
of Sciences, Yunnan 650224).
2.3. Experimental procedures
2.3.1. Syntheses of compounds and structural analyses
N-Hydroxylsuccinamide biotinyl ester (NHS-Biotin) was pre-
pared and purified as before [35]. Mono-dansylated ethylenedi-
amine (DEDA) was prepared via the reaction of ethylenediamine
in great excess with dansyl chloride (30:1) in dichloromethane,
washed repeatedly with large volumes of water and 0.3% HCl, fol-
lowed by purification via silica gel chromatography. Reaction of
NHS-biotin with each indicated alkyl amine in dichloromethane
at room temperature for 24 h gave the desired biotinyl amide
(Scheme 2); each intended biotinyl amide, after the removal of
most solvents under reduced pressure at 40 ◦C, was precipitated
and washed with ethyl ether, purified by silica gel chromatogra-
phy via the elution with 7% methanol in chloroform. Methyl biotin
ester (BME) was prepared with NHS-biotin in methanol at 40 ◦C for
72 h, followed by removal of methanol, wash with ethyl ether and
column chromatography on silica gel. From each column, eluted
fractions without contaminants detectable by TLC were pooled, and
solvents were removed to give each biotin derivative. Formula com-
positions were analyzed by ESI-HRMS, and NMR data in DMSO-d6
were collected at 25 ◦C (Note S1, Supplementary Material).
Scheme 2. Syntheses and structures of the used biotin derivatives.
As for BNEDA, the excitation was realized at 280 nm to measure the
fluorescence of the bound BNEDA at 430 nm or SAV at 340 nm. As
for BDEDA, the excitation was made at 280 nm to measure SAV flu-
orescence at 340 nm, but at 285 nm to measure the fluorescence of
the bound BDEDA at 525 nm (the half-frequency scattering of the
excitation light was reduced). With BNEDA varying from 0.18 ␮M
to 0.54 ␮M as indicated, the final levels of SAV ranged from 0.10 ␮M
to 0.30 ␮M to ensure competitive binding, correspondingly. With
BDEDA at 0.50 ␮M as the reference ligand, the final level of SAV was
0.30 ␮M, unless otherwise stated. The final level of biotin was below
60.0 ␮M and that of any biotin derivative was below 12.0 ␮M to give
final contents of organic solvents below 0.4% for negligible inter-
ference with fluorescence of components in reaction mixtures. The
2.3.2. Binding reaction and measurement of fluorescence
BNEDA was calibrated with its absorptivity of 5.3 mM−1 cm−1
at 325 nm, and BDEDA was determined with its absorptivity of
4.4 mM−1 cm−1 at 338 nm (Supplementary Material). A stock solu-
tion of biotin at 20.0 mM was made in dimethylsulfone and stock
solutions of other biotin derivatives at 4.0 mM were prepared in
methanol. The amount of binding sites on SAV was titrated with
BNEDA and the buffer was 0.10 M sodium phosphate at pH 7.0 as
before [35]. Reaction mixtures in total of 3.0 mL at 25 ◦C contained
100 ␮L BNEDA (or BDEDA) diluted by the buffer, 100 ␮L SAV solu-
tion and 200 ␮L solution of biotin (derivative) diluted by the buffer.

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Y. Xie et al. / Spectrochimica Acta Part A 77 (2010) 869–876
binding reaction was initiated by the addition of SAV. Three min-
utes after the addition of SAV, the fluorescence of reaction mixtures
was measured within 5 min. Dissociation constants of BDEDA was
estimated via competitive binding against biotin [31,35].
Eq. (1) is negligible. Namely, Eq. (4) should apply.
CFProt
CTProt
KdR
≤
≤ 0.02
(4)
CTRef − CTProt
CTRef is usually restricted within 5 ␮M for the linear response
of FX. Consequently, Eq. (4) can be easily validated with KdR of
a fluorescent reference ligand below 0.1 ␮M and CTProt at about
0.1 ␮M. With KdR of a fluorescent reference ligand over 0.1 ␮M,
the contribution of FFProt should be included to approximate BR
with inevitable complexity. With negligible FFProt, the fluorescence
of reaction mixture at 340 nm under the excitation at 280 nm is
approximated as Eq. (5).
2.4. Data processing
Details to approximate binding ratio (BR) of any fluorescent ref-
erence ligand as the FRET acceptor and to estimate the dissociation
constant (KdX) of any nonfluorescent candidate ligand were given
in Section 3. Experimental results were in mean standard devia-
tion (SD). Comparison was made by Student’s t-test with P < 0.05
for significant difference.
FX ≈ SBCan CBCan + SBRef CBRef
(5)
The sum of CBRef and CBCan is equal to CTProt if CFProt is negligible,
and thus Eq. (6) applies.
3. Chemometrics for calculating ligand affinities
To simplify the approximation of the binding ratio of a reference
ligand, the background fluorescence is corrected for a negligible
intercept of linear response with each interested component in
the following reaction mixtures. For reaction mixtures contain-
ing a protein at a constant level indexed as its binding site(s)
SBCan CTProt − FX ≈ (SBCan − SBRef)CBRef
(6)
Assigning SBRef CTProt to Fmin, which accounts for the fluores-
cence of all the protein that is bound by the reference ligand, and
SBCan CTProt to Fmax, which accounts for the fluorescence of all the
(CTProt), a fluorescent reference ligand at a constant level (CTRef
)
protein that is bound by the candidate ligand, there is Eq. (7).
and a candidate ligand at varying levels for competitive binding,
the fluorescence (FX) is the sum of those from interested com-
ponents including the complex of the protein with the reference
ligand (FBRef), the complex of the protein with the candidate ligand
(FBCan), the free reference ligand (FFRef), the free protein (FFProt) and
the free candidate ligand (FFCan). Obviously, to ensure competitive
binding, CTRef should be reasonably larger than CTProt, and Eq. (1)
applies as long as FX is within its linear range and there are no inter-
actions among aforementioned interested components to quench
their fluorescence each other.
Fmax − FX
CBRef
≈
(7)
SBCan − SBRef
For the maximal binding of the reference ligand to the protein,
CBRef can be taken as CTProt. With BR defined as the percentage of
CBRef to CTProt, Eq. (8) applies.
CBRef
Fmax − F
Fmax − FX
CTProt(SBCan −XS
Fmax − Fmin
BR =
≈
=
)
(8)
CTProt
BRef
Fmin and Fmax can be determined via titration, or approximated
as follows as long as FFProt is negligible. (a) Fmax can be approx-
imated as the fluorescence of a reaction mixture containing the
protein at CTProt and a candidate ligand at a suitable level to bind all
the protein as long as the candidate ligand in excess does not affect
Fmax. (b) Fmin can be approximated as the fluorescence of a reaction
mixture containing the protein at CTProt and the reference ligand
at a suitable level to bind all the protein as long as the reference
FX = FBCan + FBRef + FFRef + FFProt + FFCan
(1)
Assume that the fluorescence of each interested component
responds linearly to its concentrations with a negligible intercept.
Namely, FBRef, FBCan, FFRef, FFProt and FFCan are directly propor-
tional to their concentrations (CBRef, CBCan, CFRef, CFProt and CFCan
,
respectively) with slopes of SBRef, SBCan, SFRef, SFProt and SFCan, cor-
respondingly. Therefore, Eq. (2) applies.
ligand in excess doesn’t affect Fmin
.
FX = SBCan CBCan+SBRef CBRef+SFRef CFRef+SFProt CFProt+SFCan CFCan (2)
3.2. Approximation of BR from fluorescence of a reference ligand
Upon the excitation around 280 nm, both protein fluorescence
around 340 nm and the characteristic fluorescence of the bound
reference ligand will be altered via FRET during competitive bind-
ing, and BR is approximated with different fluorescence signals as
follows.
With any suitable reference ligand that has large enough Stokes
shift as the FRET acceptor when tyrosine/tryptophan residues act
as the donors, the protein by the excitation around 280 nm should
have negligible fluorescence at the wavelength to quantify the
characteristic fluorescence of the bound reference ligand. At such
a detection wavelength under excitation around 280 nm, free or
bound nonfluorescent candidate ligands should also have negligi-
ble signals. Therefore, by quantifying the fluorescence of the bound
reference ligand upon the excitation around 280 nm, FX princi-
pally originates from the bound and the free reference ligand as
described in Eq. (9).
3.1. Approximation of BR from protein fluorescence
With protein fluorescence around 340 nm to approximate BR,
nonfluorescent candidate ligands of asymmetric structures and any
reference ligand within limited concentration ranges should have
negligible fluorescence at 340 nm. If the concentration of the free
protein, CFProt, is negligible with respect to CTProt during compet-
itive binding, the contribution of FFProt to FX could be neglected.
FX ≈ SBRef CBRef + SFRef CFRef
(9)
Eq. (9) is independent on KdR. Assigning SFRef CTRef to Fmin and
SBRef CTProt to Fmax, Eq. (10) applies because CTRef is a constant [35].
Consequently, Eq. (11) applies.
Assigning the dissociation constant of the reference ligand to KdR
there is Eq. (3).
,
CBRef
CFRef
CTProt
CFProt
=
× KdR
≤
× KdR
(3)
FX − Fmin ≈ (SBRef − SFRef)CBRef
FX − Fmin
SBRef − SFRef
(10)
(11)
CTRef − CTProt
If CTRef minus CTProt is 50 times higher than KdR, CFProt is negli-
gible with respect to CTProt so that the contribution of FFProt to FX in
CBRef
≈

Y. Xie et al. / Spectrochimica Acta Part A 77 (2010) 869–876
873
Similar to the derivation of BR from protein fluorescence at
340 nm, there is Eq. (12) from the fluorescence of the bound ref-
erence ligand under the excitation around 280 nm.
CBRef
FX − Fmin
FX − Fmin
BR =
≈
=
(12)
CTProt
(SBRef − SFRef)CTProt
Fmax − SFRef CTProt
Fmin for the reference ligand at CTRef is directly determined by
experimentation. Fmax with the protein at CTProt is preferred to be
determined via titration with the reference ligand. With a reference
ligand of KdR below nanomolar, Fmax can be approximated as the
fluorescence of a reaction mixture containing the protein at CTProt
and the reference ligand in slight excess after the effect of the free
reference ligand in excess is corrected. SFRef CTProt is calculated from
SFRef and CTProt or be approximated as Fmin if CTRef is just 10% in
excess to CTProt. So, Eq. (13) applies.
CBRef
FX − F
Fmax − mF in
BR =
≈
(13)
CTProt
min
Fig. 1. Excitation spectra of BNEDA, SAV and their complexes detected at 450 nm.
(a) BNEDA alone; (b) SAV alone; (c) 0.1 ␮M SAV + 0.12 ␮M BNEDA; (d) 0.1 ␮M
SAV + 0.12 ␮M BNEDA + 0.4 ␮M biotin; (e) 0.1 ␮M SAV + 0.12 ␮M BNEDA + 2.4 ␮M
biotin.
3.3. Estimation of dissociation constant of a candidate ligand
KdR can be estimated via competitive binding of a reference lig-
and against a nonfluorescent ligand with a known affinity, or be
determined via titration when applicable. EC50 of any candidate
ligand was determined from regression analysis of the linear part
of the plot of BR to logarithmic concentrations of a nonfluorescent
candidate ligand during competitive binding. Assigning the disso-
ciation constant of a candidate ligand to KdX, the depletion of the
reference ligand during competitive binding should be considered
to derive KdX and there was Eq. (14) [29,30].
ligand and the quantification of either fluorescence signal. (c) With
a protein of multiple binding sites, deviations are expected if candi-
date ligands quench excited states of tyrosine/tryptophan resides
in adjacent binding sites or the fluorescence of the acceptor bound
in adjacent sites. (d) Upon the excitation around 280 nm, if any can-
didate ligand has obvious signals at the wavelength to quantify the
fluorescence of the bound reference ligand, it is prone to cause the
deviation in KdX if the quantum yield of fluorescence of the bound
reference ligand is relatively small.
KdX
KdR
2EC50
2CTRef − CTProt − 2KdR
CTProt
=
−
(14)
2CTRef − CTProt
4. Experimental results
In competitive binding systems, (2CTRef − CTProt) was much over
nanomolar while KdR for either BNEDA or BDEDA was below pico-
molar [31,35–38]. Thus, Eq. (15) applied.
Fluorescent biotin derivatives with KdR below 10 nM could be
easily designed [35–38]. With tryptophan residues as intrinsic
donors, both dansylamide and naphthylamine are effective accep-
tors [35]. But dansylamide has a quantum yield of fluorescence in
aqueous solutions lower than that of naphthylamine. Therefore, to
SAV, BNEDAandBDEDAwere usedasthe reference ligands of differ-
ent quantum yields to cross-validate this homogenous competitive
assay.
KdX
KdR
2EC50 − CTProt
2CTRef − CTProt
≈
(15)
3.4. Optimization and expected properties
For this new homogenous assay of ligand affinities, the design
of suitable fluorescent reference ligands as the FRET acceptors is
already discussed [35]. To enhance this homogeneous competitive
assay, the following points should be considered. (a) The binding of
the reference ligand should quench protein fluorescence as effec-
tively as possible but the binding of any nonfluorescent candidate
ligand should quench fluorescence of the protein as negligibly as
possible. (b) Due to the potential quench of fluorescence of com-
plexes by a reference ligand or candidate ligand in great excess,
Fmin and Fmax in both Eq. (8) and Eq. (12) should be checked for
their validity. (c) The validity of Eq. (8) requires the affinity of a
suitable reference ligand as high as possible. The validity of Eq. (12)
requires the quantum yield of fluorescence of a suitable reference
ligand as high as possible. When a fluorescent reference ligand as
the FRET acceptor has an affinity strong enough to validate Eq. (8)
and simultaneously a high enough quantum yield of fluorescence
in complexes with the protein, both Eq. (8) and Eq. (12) can be used.
The following properties are expected and can be used for cross-
validating this new homogeneous competitive assay. (a) If both Eq.
(8) and Eq. (12) are validated with a reference ligand, consistent KdX
of a nonfluorescent candidate ligand could be obtained by quanti-
fying the fluorescence of the protein and the fluorescence of the
bound reference ligand. (b) If both Eq. (8) and Eq. (12) are validated
with two reference ligands, KdX of a nonfluorescent candidate lig-
and should be consistent, regardless of the use of either reference
4.1. Characterization of FRET in complexes
BNEDA and BDEDA had their absorption valleys around 280 nm
and 285 nm, and absorption peaks around 325 nm and 340 nm,
respectively (Figure S1, Supplementary Material). Upon direct exci-
tation of free BNEDA and free BDEDA at 325 nm and 340 nm,
their characteristic emission peaks appeared around 450 nm and
515 nm, respectively, but quantum yield of fluorescence of BDEDA
was less than 10% of that of BNEDA. Meanwhile, they showed
excitation valleys around 280 nm. After the binding to SAV, exci-
tation of bound BNEDA at 325 nm and bound BDEDA at 340 nm
produced their characteristic emission peaks around 430 nm and
525 nm, respectively, but the bound BDEDA still showed much
lower quantum yield of fluorescence at 525 nm than that of the
bound BNEDA at 430 nm. By excitation of the complexes at 280 nm,
there was about 70% reduction of SAV fluorescence at 340 nm by
the bound BNEDA or BDEDA and meanwhile there was the charac-
teristic emission of the bound reference ligand. Upon competitive
binding against biotin, the excitation peak at 280 nm of SAV to pro-
duce fluorescence at 340 nm was rescued but the fluorescence peak
at 430 nm with BNEDA or that at 525 nm with BDEDA was reduced,
in a manner dependent on biotin concentrations (Figs. 1 and 2,
Figure S2, Supplementary Material).

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Y. Xie et al. / Spectrochimica Acta Part A 77 (2010) 869–876
Fig. 2. . Emission spectra of BDEDA, SAV and their complexes excited at 280 nm.
Solutions were completely the same as those in Fig. 1.
In solutions with free tryptophan and free BDEDA at levels com-
parable to those in solutions used above, there were no detectable
fluorescence peaks around 525 nm by excitation at 285 nm. So was
it with free tryptophan and free BNEDA in solutions by excitation at
280 nm. And NEDA or DEDA at the same level had no effects on SAV
fluorescence. In solutions of free tryptophan and free BDEDA (or
free BNEDA), averaged distances between the FRET pair were over
15 nm [35]. But such distances in the complex of SAV and BDEDA
or BNEDA were within 5.0 nm (Scheme 1). Sensitivity to distances
between a donor and acceptor was the nature of FRET [6]. Therefore,
FRET in complexes of SAV with BDEDA or BNEDA should principally
account for the quench of SAV fluorescence and the characteristic
fluorescence of the bound BNEDA or BDEDA.
Fig. 3. Estimation of the affinity of BNEDA via competitive binding against biotin.
(a) With fluorescence of bound BNEDA at 430 nm under the excitation at 280 nm.
(b) With fluorescence of SAV at 340 nm under the excitation at 280 nm.
(n = 4) with fluorescence at 525 nm under excitation at 285 nm
(Fig. 4a).
Biotin at 20 ␮M quenched SAV fluorescence by only about 10%,
via mechanisms other than FRET. By measuring SAV fluorescence at
4.2. Dissociation constants of reference ligands
By quantifying the fluorescence of a bound reference ligand, its
KdR could be estimated based on its competitive binding against
biotin. (a) For BNEDA, Fmax at 430 nm upon the excitation at 280 nm
was estimated with BNEDA in 10% excess to SAV. When the effect
of free BNEDA on Fmax was corrected, paired t-test gave statistically
lower KdR of BNEDA by Eq. (12) but the difference was within 3%
(Table 1). Averaged KdR of BNEDA was (14 2) fM (n = 12) (Fig. 3a).
When concentrations of SAV and BNEDA were varied over wide
ranges, BNEDA had consistent KdR by Eq. (12). (b) Free BDEDA had
negligible effects on Fmax. KdR of BDEDA by Eq. (12) was (26 2) fM
Table 1
Dissociation constants of BNEDA and BDEDA based on their competitive binding
against biotin. Number in parenthesis was that for independent assays. Other details
were described in Section 2.3.
Compound
Conditions(␮M)
KdR (fM)
Cprot
CTRef
Emission via FRET
Quench via FRET
BNEDA
0.30
0.30
0.50
0.35
0.18
0.10
15 3 (5)
12 2 (2)
13 2 (3)
14 2 (2)
33 4 (4)a
14 2 (2)b
c
0.10
–
0.025d
18 2 (2)
BDEDA
0.30
0.50
26 2 (4)
28 3 (4)
a
Significantly higher than that of BNEDA under any other condition, but all other
results for BNEDA had no differences by Student’s two-way t-test.
b
Fmin was from titration, which was significant higher than that at 0.50 ␮M
Fig. 4. Estimation of the affinity of BDEDA via competitive binding against biotin.
(a) With fluorescence of bound BDEDA at 525 nm under the excitation at 285 nm.
(b) With fluorescence of SAV at 340 nm under the excitation at 280 nm.
BNEDA.
c
Not determined due to low sensitivity on Shimadzu RF540.
d
Fluorescence was monitored on Shimadzu RF5301PC.

Y. Xie et al. / Spectrochimica Acta Part A 77 (2010) 869–876
875
Table 2
for such deviations. (c) BCHA had some scattering signals over
460 nm. By quantifying the fluorescence at 525 nm of the bound
BDEDA that had low quantum yield of fluorescence, the scatter-
ing signal of BCHA caused positive deviation to its KdX by Eq. (12)
(Figure S4, Supplementary Material). By quantifying SAV fluores-
cence at 340 nm with BDEDA as a reference ligand, KdX of BCHA by
Eq. (8) was consistent with that via Eq. (12) by using BNEDA as the
reference ligand (Table 2).
Dissociation constants of some nonfluorescent biotin derivatives with either BNEDA
or BDEDA as the reference ligand. Each result was the average in duplicate plus
standard deviation. All the fluorescence was monitored on Shimadzu RF540.
Compound
KdX (fM, BNEDA)a
Kdx (fM, BNEDA)b
Kdx (fM, BDEDA)c
BCHA
BMPL
BDETA
BME
BETA
BBZA
28
82
3
3
6
2
5
2
29
84
2
1
6
2
3
2
30
83
3
3
8
3
6
2
188
43
193
45
163
41
94
92
87
42
41
44
5. Discussion and conclusion
a
Fluorescence was monitored at 430 nm by the excitation at 280 nm with BNEDA
The experimental results described above clearly supported our
theoretical considerations on this new homogeneous competitive
assay of ligand affinities. Other mechanisms may also quench the
fluorescence of proteins upon the binding of fluorescent reference
ligands. In fact, Eq. (8) only requires the quench of protein fluo-
rescence, and Eq. (12) only requires the transfer of energy from
excited tyrosine/tryptophan residues, upon the binding of a fluores-
cent reference ligand. Hence, this homogeneous competitive assay
of ligand affinities should be effective even if other mechanisms
also account partly for the quench of protein fluorescence.
This new homogenous competitive assay requires a suitable
fluorescent reference ligand as the FRET acceptor to tyro-
sine/tryptophan residues as intrinsic donors. The general guideline
to design such FRET acceptors to common proteins was proposed
previously [35]. Typically, this new homogenous competitive assay
of ligand affinities should be applicable to the following three
types of proteins. (1) Proteins that bind macromolecular ligands,
including protein kinases, signaling proteins, receptors of cytokines
and DNA polymerases, all of which are important drug targets.
(b) Proteins that possess binding sites large enough to accommo-
date nucleotides including ATP, GTP, cAMP, or cGMP. (c) Proteins
such as SAV and albumin whose putative ligands have some
groups exposed to solvents, on which suitable fluorophores can be
attached to act as acceptors.
as the reference ligand, by presetting CTRef = 0.18 ␮M, CProt = 0.10 ␮M, and KdR = 14 fM.
b
Fluorescence was monitored at 340 nm by the excitation at 280 nm with BNEDA
as the reference ligand, by presetting CTRef = 0.50 ␮M, CProt = 0.30 ␮M, and KdR = 33 fM.
c
Fluorescence was monitored at 340 nm by the excitation at 280 nm with BDEDA
as the reference ligand, by presetting CTRef = 0.50 ␮M, CProt = 0.30 ␮M, and KdR = 27 fM.
340 nm under excitation at 280 nm, affinities of BNEDA and BDEDA
were also estimated via competitive binding against biotin. (a)
BNEDA over 0.35 ␮M reduced the approximated Fmin at 340 nm
in a concentration-dependent manner. When Fmin at 340 nm with
BNEDA below 0.35 ␮M were used in Eq. (8), KdR of BNEDA was
consistent with that by using fluorescence of the bound BNEDA at
430 nm (Fig. 3b). When Fmin at 340 nm approximated with BNEDA
at 0.50 ␮M was used in Eq. (8), KdR of BNEDA showed significant
positive deviation from that by using its fluorescence at 430 nm
(Table 1). (b) BDEDA had no effects on Fmin at 340 nm. With SAV flu-
orescence at 340 nm, KdR of BDEDA was consistent with that using
its fluorescence at 525 nm (Fig. 4b).
The overall coefficients of variations were about 10%, supporting
reasonable reproducibility of this new homogenous competitive
assay of ligand affinities based on estimation of EC50
.
4.3. Dissociation constants of some nonfluorescent biotin
derivatives
Taken together, based on the cost, efficiency, sensitivity, alter-
ation on protein functions and the suitability for routine practice of
HTS, this homogeneous competitive assay of ligand affinities can be
an advantageous alternative of the classical homogenous competi-
tive assay of ligand affinities when fluorescent reference ligands are
available as the suitable FRET acceptors while tyrosine/tryptophan
residues in a protein as the intrinsic donors.
Low quantum yield of fluorescence of BDEDA at 525 nm made
BDEDA only suitable to approximate BR via Eq. (8). By using BNEDA
to quantify its fluorescence at 430 nm, KdX of each tested nonflu-
orescent biotin derivative via Eq. (12) was consistent with that
via Eq. (8) using BDEDA as the reference ligand to quantify SAV
fluorescence at 340 nm (Table 2).
By using BNEDA as the reference ligand at 0.50 ␮M without cor-
rection of the effect of BNEDA in excess on Fmin at 340 nm, KdX
of each tested nonfluorescent biotin derivative by Eq. (8) was still
consistent with that by using Eq. (13) with the characteristic fluo-
rescence of the bound BNEDA at 430 nm, when KdR of BNEDA under
the corresponding conditions was used (Table 2). Affinity of each
tested nonfluorescent biotin derivative relative to that of biotin, by
using Eq. (8) and parameters with BNEDA at 0.50 ␮M to calculate
KdR and KdX, was consistent with that by using Eq. (8) and BDEDA
as the reference ligand (Table S1, Supplementary Material).
Acknowledgements
This work was supported by Program for New Century Excel-
lent Talents in University (NCET-10, the Education Ministry of
China), the National Natural Science Foundation of China (no.
30472139)andChongqingMedicalUniversity(no. CX200203). Miss
Ying Zhang currently worked in Changlong Enterprise Group, LTD,
Nanping, 400060 Chongqing, China.
Appendix A. Supplementary data
4.4. Deviations under special situations
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.saa.2010.08.021.
Deviations were observed as follows. (a) By using BNEDA as
the reference ligand to monitor its characteristic fluorescence at
430 nm, EC50 of BDEDA as a candidate ligand was so small that a
negative KdX of no physical meaning was observed (Figure S3, Sup-
plementary Material). The quench of excited tryptophan residues
in adjacent site, where BNEDA was bound, may account for neg-
ative KdX of BDEDA with BNEDA as the reference ligand [35]. (b)
When BDEDA was used as the reference ligand, the fluorescence at
340 nm showed negligible responses to increasing concentrations
of BNEDA as a candidate ligand. The comparable quenching effects
of BNEDA and BDEDA on SAV fluorescence at 340 nm could account
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