evaluated (i) the general effect of polarity environment
which is predicted to decrease upon binding from aqueous
determined after smoothing by averaging over 10 adjacent
points. The solvent effects were evaluated by plotting of
(
solvent to protein) and (ii) the influence of amino acids
reactive groups. We demonstrated that precise analysis of
the fluorescence spectra can provide valuable structural
information about the SG/enzyme interaction and we were able
nꢀ max vs. e (relative static permittivity), using the Lippert plot
r
2
( nꢀ ex À nꢀ ) vs. Df, where Df = (e À 1)/(2e + 1) À (n À 1)/
em
17
(2n + 1), or the Kamlet–Taft solvatochromic analysis:
r
r
2
18,19
+
+
+
V = nꢀ
0
+ aa + bb + pp*
(2)
to identify the SG -binding site on the Na /K -ATPase.
where V is the evaluated spectral characteristic (in our case nꢀ ex
,
nꢀ em or nꢀ ex- nꢀ em), nꢀ
, a, b and p are the fitted parameters, the
0
Experimental
values of a (describing the ability of the solvent to donate a
proton in a solvent-to-solute hydrogen bond), b (describing
the ability of the solvent to accept a proton in a solute-to-
solvent hydrogen bond), and p* (which measures the ability of
the solvent to stabilize a charge or a dipole by virtue of its
dielectric effect) for individual solvents were taken from
ref. 18. Chloroform as a chlorinated solvent was excluded
from the Kamlet–Taft analyses.
Chemicals
Sanguinarine (13-methyl[1,3]benzodioxolo[5,6-c]-1,3-dioxolo-
[
4,5-i]phenanthridinium chloride) and NADH were purchased
from Sigma-Aldrich (Prague, Czech Republic). Dihydrosan-
guinarine (13,14-dihydro-13-methyl[1,3]benzodioxolo[5,6-c]-
1
,3-dioxolo[4,5-i]phenanthridine, DHSG), 99% purity, MP
1
89–191 1C was prepared from sanguinarine by reaction with
NaBH in methanol.
After elimination of data revealing apparent specific
+
reactivity between solvent and SGOH, SG or DHSG, the
1
6
4
+
Effect of polarity on the fluorescence spectra of SG ,
SGOH and DHSG was evaluated in chloroform, butanol,
acetone, ethanol, methanol, DMSO, 2-butanethiol, 1,2-ethane-
dithiol, and aqueous Tris buffer solution, pH 7.5. All the
solvents were from Sigma and were of spectrophotometric
grade.
remaining points in the graphs were fitted by linear function.
Emission spectra of 1.25 mM SG were also measured in the
+
+
presence of 5 mM isolated porcine cerebral cortex Na /K -
ATPase (Sigma), in the 10 mM Tris buffer, pH 7.5, containing
1
0 mM NaCl and 140 mM KCl and 20 mM sucrose.
Neutral amino acid analogues Ac-Ser-OMe, Ac-Tyr-NH2,
Ac-Glu-NH , Ac-Gln-NH , Ac-Lys-NH ÁHCl, Ac-Arg-NH -
Results
2
2
2
2
acetate salt, where Ac stands for acetyl and Me for methyl,
were supplied by Bachem and Ac-Cys-OMe was supplied
by Fluka.
12
As we had shown before SGOH in aqueous buffer at pH 7.5
exhibits maxima of excitation/emission spectra at 327/418 nm
À1
which corresponds to 30 510/23 810 cm on the wavenumber
+
scale (ESI, Fig. S1w), while SG exhibits spectra with maxima
at 475/590 nm which corresponds to 21 030/16 800 cm on the
Excitation and emission spectra
À1
+
wavenumber scale. Note that the SG excitation spectrum is
The steady-state fluorescence emission spectra of 10 mM SG or
5
mM DHSG were measured on a spectrofluorimeter F4500
Hitachi, Japan). Data were collected using excitation
apparently composed of two strongly overlapping peaks
(
(ESI, Fig. S2w). The DHSG spectra have maxima at 327/446
À1
wavelength 327 nm or 475 nm for SG, and 327 nm for DHSG,
À1
nm, which corresponds to 30 510/22 350 cm (ESI, Fig. S3w).
with a scan-speed 240 nm min . Slits were set to 10 nm for
The spectra are essentially the same in distilled water, Tris or
phosphate buffer (not shown). The spectra were reproducible
with accuracy of Æ 2 nm, which corresponds to approx.
both the excitation and emission channel, respectively. Spectra
were collected in 0.2 nm steps and measurements were
performed at 295 K.
À1
À1
200 cm in the 327 nm region, 100 cm in the 418 nm, in
À1
The spectra were measured in 100% solutions of
chloroform, butanol, acetone, ethyl alcohol, methanol,
DMSO, 1-butanethiol, 1,2-ethanedithiol and aqueous buffer
solution (pH 7.5) and 10% solution of 1-butanethiol and 1,2-
ethanedithiol in 20 mM Tris buffer, pH 7.5. Further, a 1 mM
solution of the neutral analogs of amino acids Ser, Tyr, Glu,
Gln, Lys, Arg and Cys in Tris buffer, pH 7.5 were used to test
the influence of the functional groups found within proteins.
The measurement was performed in freshly prepared solutions
of amino acid analogs. The experiment with Cys analog was
done in a buffer that was deoxygenized by nitrogen to avoid
oxidation.
the 446 nm, and in the 475 nm regions, and 50 cm in the
5
90 nm region.
Solvent effects
The spectral changes observed in various solvents can be
attributed either to general changes in fluorophor environment
polarity or to the specific interactions between the fluorophor
and reactive groups of the solvent molecules.
For SGOH, we can see little red-shift (compared to the
aqueous Tris buffer, pH 7.5) in the emission spectrum
À1
obtained in the 1-butanethiol (À870 cm ) and excitation
À1
spectrum in 1,2-ethanedithiol (À1110 cm ). Notably,
Solvent effects are usually evaluated in the wavenumber
scale. Therefore, the spectra were converted into the wave-
number scale using:
these changes were not observed in the 10% solution of
1
-butanethiol in the aqueous Tris buffer. Evaluation of the
dependence of both excitation and emission peaks on the
relative permittivity of the solution (Fig. 2), as well as a
Lippert plot (Fig. 5), showed no dependence of the fluores-
cence properties on environment polarity (see Table 1).
Detailed analysis using the Kamlet–Taft parameters that also
2
I( nꢀ ) = I(l)Ál
(1)
where l is wavelength, nꢀ is corresponding wavenumber and I is
fluorescence intensity. The maximum of the peak nꢀ max was
1
1336 Phys. Chem. Chem. Phys., 2010, 12, 11335–11341
This journal is c the Owner Societies 2010
Janovska, Marika
