X. Dang et al. / Carbohydrate Research 346 (2011) 949–955
951
(75%): 1H NMR (400 MHz, CDCl3): d 7.86 (1H, d, J = 7.6 Hz, H-1),
2.6. Calculation of binding parameters
7.69 (1H, t, J = 8.0 Hz, H-2), 7.27 (1H, d, J = 8.4 Hz, H-3), 5.40 (1H,
d, J = 3.0 Hz, H-10), 5.10 (1H, br, H-7), 4.92 (1H, J = 2.6 Hz, H-100),
4.07 (1H, m, H-50), 3.97 (3H, s, OMe-4), 3.86 (1H, m, H-500), 3.50
(1H, br, H-40), 3.07–3.05 (2H, m, Ha-10, H-30), 2.94 (1H, d,
J = 79.6 Hz, H-400), 2.79 (1H, d, J = 18.7 Hz, Hb-10), 2.34 (3H, s, H-
14), 2.16–2.14 (2H, m, Ha-200, Ha-8), 2.01–1.99 (1H, m, Hb-8),
1.82–1.78 (1H, m, Hb-200), 1.68–1.66 (2H, m, H-20), 1.25–1.21 (9H,
m, H-60, H-600, CH3-300). HRESIMS (positive-ion mode): calcd for
The binding constants can be found from the static quenching
equation:
ðF0 ꢁ FÞꢁ1 ¼ F0ꢁ1 þ Kꢁ1F0ꢁ1½Qꢃꢁ1
;
ð4Þ
where K (intercept/slope) denotes the binding constant of
a1 and
the biomolecule, which can be calculated from the slope and inter-
cept of Lineweaver–Burk curves, F0 and F are the fluorescence inten-
sities without and with quencher, and [Q] is the quenching rate
constant of the biomolecule.
C
34H41NO13Na+, m/z 694.2470, found, m/z 694.2463.
If the enthalpy change (DH) does not vary significantly over the
2.4. Measurements of the fluorescence spectrum
temperature range studied, then its value and that of
D
S can be
determined from the Van’t Hoff equation:14
Under the optimum physiological conditions described above,
2.0 mL Tris–HCl buffer solution, 2.0 mL NaCl solution, appropriate
amounts of HSA, and a1 were added into a standard flask and di-
ln K ¼ ꢁ
D
H=RT þ
DS=R:
ð5Þ
In Eq. 5, K is the binding constant at corresponding temperature
and R is the gas constant. The enthalpy change ( H) and the entro-
py change ( S) are calculated from the slope and ordinate of the
luted to 10.0 mL with double-distilled water. Fluorescence quench-
ing spectra of HSA were obtained at an excitation wavelength of
280 nm and an emission wavelength of 300–450 nm. Fluorescence
spectra in the presence of other ions were also measured under the
same conditions. In addition, the UV absorption and synchronous
fluorescence spectra of system were recorded.
D
D
Van’t Hoff relationship. The free-energy change (
from the following relationship:
D
G) is estimated
D
G ¼
D
H ꢁ T S ¼ ꢁRT ln K:
D
ð6Þ
2.5. Principles of fluorescence quenching
2.7. Characteristics of synchronous fluorescence method
The fluorescence intensity of a compound can be decreased by a
variety of molecular interactions that include the following: ex-
cited-state reactions, molecular rearrangement, energy transfer,
ground-state complex formation, and collisional quenching.12
Fluorescence quenching is described by the Stern–Volmer equation
(Eq. 1):
The synchronous fluorescence spectra were obtained by simul-
taneously scanning the excitation and emission monochromators.
Thus, the synchronous fluorescence applied to the equation of syn-
chronous luminescence:15
F ¼ kcdEexðkem
ꢁ
D
kÞEemðkemÞ;
ð7Þ
F0=F ¼ 1 þ Kqs0½Qꢃ ¼ 1 þ Ksv½Qꢃ;
ð1Þ
where F is the relative intensity of synchronous fluorescence,
D
k =
D
kem ꢁ kex is a constant, Eex is the excitation function at the gi-
where F0 and F are the fluorescence intensities before and after the
addition of the quencher, respectively. Kq, KSV 0, and [Q] are the
quenching rate constant of the biomolecule, the Stern–Volmer dy-
ven excitation wavelength, Eem is the normal emission function at
the corresponding emission wavelength, c is the analytical concen-
tration, d is the thickness of the sample cell, and k is the character-
istic constant comprising the ‘instrumental geometry factor’ and
related parameters. Since the relationship of the synchronous fluo-
, s
namic quenching constant, the average lifetime of the biomolecule
without quencher
(s
0 = 10ꢁ8 s), and the concentration of the
quencher, respectively. The concentration of the quencher should
be the free ligand concentration, but it is not known in the experi-
ment. So, in our analysis it was approximated by the total concen-
tration of the quencher. For higher ligand concentrations, in excess
of available specific protein binding sites, this approximation is va-
lid. Obviously,
rescence intensity (F) and the concentration of
F equation, F should be in direct proportion to the concentration of
1.
The optimal values of the wavelength intervals (
tant for the correct analysis and interpretation of the binding
mechanism. When the wavelength interval ( k) is fixed at 60 nm
a1 should follow the
a
D
k) are impor-
D
Kq ¼ Ksv
=s0
:
ð2Þ
of protein, the synchronous fluorescence has the same intensity
as the emission fluorescence following excitation at 280 nm; only
the emission maximum wavelength and shape of the peaks were
changed.16–18 Thus, these synchronous fluorescence measure-
ments can be applied to calculate association constants similar to
the emission fluorescence measurements. Therefore, the synchro-
nous fluorescence measurements can deduce the binding mecha-
nism as the emission fluorescence measurements did. In this
study, the synchronous fluorescence spectra of tyrosine residues
and tryptophan residues were measured at kem = 280 nm
Hence, Eq. 1 is applied to determine KSV by linear regression of a
plot of F0/F against [Q]. In many instances, the fluorophore can be
quenched both by collision and by complex formation with the
same quencher. In these cases, the Stern–Volmer plot exhibits an
upward curvature, concave toward the y-axis at high [Q], and F0/
F is related to [Q] by the following form of the Stern–Volmer
equation:13
F0=F ¼ ð1 þ KD½QꢃÞð1 þ KS½QꢃÞ;
ð3Þ
(D
k = 15 and 60 nm) in the absence and in the presence of various
where KD and KS are the dynamic and static quenching constants,
respectively. The first factor on the right-hand side in Eq. 3 de-
scribes the ‘dynamic’ quenching, resulting from encounters of
quencher and fluorophore during the excited state, and the second
factor describes the ‘static’ quenching, that is the quenching caused
by the formation of a complex between the quencher and the
fluorophore predating the excitation. This modified form of the
Stern–Volmer equation is second order with respect to [Q], which
accounts for the upward curvature observed at high [Q] when both
static and dynamic quenching occur for the same fluorophore.
amounts of 1.
a
2.8. HSA–a1 docking study
The crystal structure of HSA in complex with R-warfarin was ta-
ken from the Brookhaven Protein Data Bank (entry codes 1h9z).
The potential of the 3D structure of HSA was assigned according
to the Amber 4.0 force field with Kollman all-atom charges. The
initial structures of all the molecules were generated by the