Bioorganic & Medicinal Chemistry Letters 22 (2012) 7256–7260

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier.com/locate/bmcl

Ferrocene labelings as inhibitors and dual electrochemical sensors of

human glutathione S-transferase P1-1
Manuel C. Martos-Maldonado, Indalecio Quesada-Soriano, Federico García-Maroto,
Antonio Vargas-Berenguel, Luís García-Fuentes ⇑
Department of Chemistry and Physics, University of Almería, Crta.de Sacramento s/n, 04120 Almería, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The inhibitory and sensor properties of two ferrocene conjugates, in which the ferrocene and glutathione
Received 11 July 2012 are linked through a spacer arm of different length and chemical structure, on human Pi glutathione
Revised 4 September 2012 S-transferase, were examined by activity assays, ITC, fluorescence spectroscopy and voltammetry. Such
Accepted 6 September 2012
ferrocene conjugates are strong competitive inhibitors of this enzyme with an enhanced binding affinity,
Available online 25 September 2012
the one bearing the longest spacer arm being the most potent inhibitor. Voltammetric measurements
showed a strong decrease of the peak current intensity and an increase of the oxidation potential upon
Keywords:
binding of ferrocene–glutathione conjugates to GST P1-1 showing that both conjugates can be used as
Inhibition
Ferrocene–glutathione conjugates
dual electrochemical sensors for GST P1-1.
Calorimetry Ó 2012 Elsevier Ltd. All rights reserved.
Voltammetry
Electrochemical sensors

A major problem which arises in the treatment of cancer with
chemotherapy is the development of resistance by the cancer cells
toward the cytostatic drug used. Such resistance is in part caused
by an increased metabolic detoxication of the drugs in the solid tu-
mors caused by enzymes such as the Pi glutathione S-transferase
(GST P1-1), the most abundant GST isoform in cancer cells.1–5 In
addition, a common feature, demonstrated in a number of different
human cancer cells, is the elevated levels of this human enzyme
relative to its concentration in the corresponding normal tissue.
Hence, this enzyme is used as biomarker.6–8 Human cytosolic GSTs
are dimeric proteins that can be grouped into at least seven gene-
independent classes (alpha, mu, pi, sigma, theta, omega, and zeta)
on the basis of their amino acid sequence.8–10 Their three-
dimensional folds do not differ significantly despite low sequence
homologies. Each subunit contains a very similar binding site
for GSH (G-site) and a second binding site for the hydrophobic
co-substrate (H-site) with structural differences in the latter
conferring some substrate selectivity.8,9,11
The development and design of GST P1-1 inhibitors and sensors
can emerge as promising therapeutic agents for managing the
development of resistance amongst anticancer agents. Further-
more, electrochemical-based protein sensors offer sensitivity and
selectivity, making them very attractive tools for protein detection.
Thus, compounds containing ferrocene (Fc) and bearing molecular
recognition binding sites have received much attention due to the
possibility of building redox-switching or sensing molecular or
supramolecular systems, which can be controlled through the
application of external stimuli.12–15 Medicinal application of ferro-
cene is also an active research area and many reports have shown
that ferrocene derivatives have a highly promising activity in vitro
against several diseases.16
In this work we have synthesized and studied the sensor prop-
erties and inhibition of a novel ferrocene glutathione conjugate
containing an acetamide moiety as a linker (FcCH2NHCOCH2SG, 1
in Scheme 1). We have evaluated the inhibitory activity and the
sensor properties of this ferrocene derivative toward recombinant
GST P1-1 and compared such properties with those obtained for a
ferrocene–glutathione analogue which does not have the acetam-
ide moiety in the spacer arm (FcCH2SG, 2 in Scheme 1). Such inter-
action studies have been carried out by activity assays, isothermal

⇑ Corresponding author. Tel.: +34 950 015618; fax: +34 950 015008.
E-mail address: lgarcia@ual.es (L. García-Fuentes). Scheme 1. Chemical structure of compounds 1 and 2.

0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.022
 M. C. Martos-Maldonado et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7256–7260 7257

titration calorimetry (ITC), spectroscopy fluorescence, and differ-
ential pulse voltammetry (DPV).
Compound 1 was synthesized in two steps starting from azido-
methyl ferrocene.17 The azide moiety of this compound was re-
duced to amine using tributylphosphine and then reacted in situ
with chloroacetic anhydride yielding the corresponding chloro-
acetamide derivative.18 This compound was conjugated with GSH
through a nucleophilic substitution of the chloride by the peptide
sulfur to obtain compound 1 (see Supplementary data for synthetic
details). The synthesis of compound 2 was carried out as
described.19
The effect of compounds 1 and 2 on the GST P1-1-catalysed con-
jugation of 1-chloro-2,4-dinitrobenzene (CDNB) to GSH was stud-
ied according to procedure of Habig & Jacoby.20,21 When dimeric
GST P1-122 was incubated with different amounts of 1, we ob-
served a stronger inhibitory effect of this ferrocene-conjugate than
that reported for 2.23 As the concentration of inhibitors was raised,
the residual GST activity markedly decreased. Thus, the IC50 values,
at 25 °C, showed a threefold increase in the inhibitory potency of 1
(6.58 lM) in comparison with that for 2 (18.7 lM). In both cases, a
competitive inhibition, towards GSH substrate, was deduced from
Lineweaver–Burk double reciprocal plots.24 The inhibitory con-
stants, Ki, were calculated from secondary plots of apparent Km,
against inhibitor concentration.25 Good straight lines were ob-
tained, and a typical example is shown in Figure 1. Based on these
Ki values, compound 1 exhibited an inhibitory activity eightfold
higher than that for 2 (Table 1).
Isothermal titration calorimetry (ITC) was also used to examine
the energetic interaction of these Fc-conjugates with the dimeric
GST P1-1 in phosphate buffer (pH 7.0) at 25 °C.26 ITC provides a di-
rect determination of the enthalpy change of binding (DH) and the
affinity constant (K), as well as the [ligand]/[protein] ratio when
the protein binding sites are fully saturated (n), which coincides
with the stoichiometry of the protein-ligand complex. The step-
wise addition of a solution of ferrocene conjugate 1 and 2 to a solu-
tion of dimeric GST P1-1 led to a series of exothermic peaks of
decreasing area. Figure 2 shows typical calorimetric thermograms
for the interaction of 1 and 2 with dimeric GST P1-1. Control exper-
iments that involve the same type of injections of conjugate solu-
tion into the same buffer were carried out in order to measure the
heat of dilution. Raw data were collected, the inhibitor heats of
dilution were corrected for and the data was integrated using the
Microcal Origin software supplied with the instrument. Calorimet-
ric data can be fitted adequately to the equal and independent
binding sites model with a 1:1 (Fc-conjugate/monomer) stoichi-
ometry, affording the thermodynamic parameters displayed in Ta-
ble 1. No evidence for ligand binding cooperativity was observed
for both Fc-conjugates and this dimeric enzyme. The competitive
behavior, towards GSH, was also re-examined by GSH displace-
ment experiments by ITC with these inhibitors.
The thermodynamic profile for both Fc-conjugates is very sim-
ilar (Table 1). The values of binding enthalpies and entropies are
in both cases negative, which is indicative that the binding process
of either inhibitor to this enzyme is enthalpically favorable and
entropically unfavorable. The binding shows a similar favourable
enthalpic term of approximately 13 kcal mol 1, which is partially
compensated by a moderate decrease in entropy ( 4.68 and
6.55 kcal mol 1 for 1 and 2, respectively). The negative Gibbs en-
ergy increases, as a consequence of the amide spacer arm between
ferrocene and glutathione moieties resulting a higher affinity of 1
with respect to 2. These results suggest that although the interac-
tion between the enzyme and 2 is enthalpically more favourable

A 25 C 3

20
2
15
KM, app
1/V

10
[I]= 0 μM 1
2 μM
5 4 μM
6 μM Ki
10 μM

0 5 10 15 20 25 -2 0 2 4 6 8 10 12
-1
1/[GSH] (mM ) [Inhibitor,1] (μM)

B 25 D
3
20

15 2
KM,app
1/V

10
[I]= 0 μM
20 μM 1
50 μM
5 80 μM Ki
120 μM

0 5 10 15 20 25 0 20 40 60 80 100 120
-1
1/[GSH] (mM ) [Inhibitor, 2] (μM)

Figure 1. Kinetic studies of GST P1-1 binding to compound 1 and 2. Double reciprocal plots for the CDNB/GSH conjugation by GST P1-1 in presence of increasing
concentrations of 1 (A) and 2 (B). Secondary plots of apparent Michaelis constants as a function of inhibitor concentration 1 (C) and 2 (D).
7258 M. C. Martos-Maldonado et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7256–7260

Table 1
Thermodynamic parameters for the ferrocene–glutathione derivatives binding to GST P1-1 in phosphate buffer (pH 7.0, 25 °C)

Compound Activity Fluorescence Calorimetry

5 1 5 1 5 1
K  10 (M ) (Ki) K  10 (M ) K  10 (M ) DG0 (kcal mol 1) DH (kcal mol 1) TDS0 (kcal mol 1
)
1 15.9 ± 0.61(0.63 lM) 9.33 ± 2.01 14.40 ± 4.45 8.37 ± 0.18 13.05 ± 0.34 4.68 ± 0.39
2 1.93 ± 0.72 (5.18 lM) 0.95 ± 0.40 2.28 ± 0.16 7.28 ± 0.04 13.83 ± 0.17 6.55 ± 0.18

Time (min)
A
0 50 100 150 200

0.0
Heat flow (µcal/s)

C
-0.5
0
-1.0

-1.5
-4

ΔHinj (kcal·mol )
-1
ΔH
B 0 -8
Heat flow (µcal/s)

-1
-12 n

-2
0 2 4 6
Molar ratio
-3
0 50 100 150 200
Time (min)

Figure 2. Isothermal titration calorimetry measurements of the binding of 1 (A) and 2 (B) to GST P1-1.Titration of 25 lM of dimeric GST P1-1 with 42–5 lL injections of
0.77 mM 1 (A) and 1.2 mM 2 (B). A preinjection of 1 lL was performed at the beginning. The area under each peak was integrated and plotted against the molar ratio of
inhibitor (1, squared symbols; 2, circles symbols) to GST P1-1 in panel C. The solid smooth line represents the best fit of the experimental data to a model with non interacting
sites. Titrations were performed in 20 mM sodium phosphate, 5 mM NaCl and 0.1 mM EDTA at pH 7 and 25 °C.

(although slightly) than that for 1, the entropic loss due to binding (DPV). DPV were performed using solutions of the conjugates
is also higher. This indicates that the thermodynamic effect of the (50 lM) prepared in 10 mM phosphate buffer (pH 7.2) with
presence of amide spacer arm in the inhibitor 1 is to slightly de- 20 mM NaCl as the supporting electrolyte.32 The differential pulse
crease the favourable enthalpy change and to increase the entropic voltammograms of 1 and 2 in the absence of GST P1-1 revealed
change due to binding. Thus, the favourable entropy change out- only one oxidation peak at 0.220 V meaning that in aqueous solu-
weighs the slight enthalpic disadvantage, resulting in an affinity tion the conjugates are present in only one electrochemical distin-
of 1 for GST P1-1 sixfold higher than that for 2. These results sug- guishable form in the time-scale of the measurement. Such
gest that a better fit of the ferrocene moiety of 1 in the hydropho- behavior is similar to that reported elsewhere.19 Both conjugates
bic H-site occurs upon complexation, as compared with conjugate showed almost the same peak current intensity at the same con-
2, possibly due to the higher length of the spacer arm between fer- centration meaning that the influence of the different chemical
rocene and GSH moieties. Moreover, additional interactions be- structure of the linkers in the diffusion coefficient is negligible
tween the linker in the case of 1 and the protein cannot be ruled since the peak current intensity is proportional to the square root
out. of the diffusion coefficient.33
Furthermore, the binding of these inhibitors to dimeric GST P1- Voltammetric measurements in the presence of increasing
1 quenches the intrinsic fluorescence of the enzyme as described amount of GST P1-1 showed a strong decrease of the peak current
elsewhere for GSH27 and other inhibitors.27–29 Fluorescence titra- intensity together with an increase in the oxidation potential
tions30 resulted in quenching curves that are consistent with the (Fig. 3). Such variation of both electrochemical parameters allows
behavior obtained by calorimetry (Table 1) (see Supplementary for the use of both ferrocene-glutathione conjugates as dual elec-
data). trochemical sensors for the detection of GST P1-1. The decrease
Therefore, the ferrocene conjugate with amide group (com- of the peak currents as the protein concentration increases is the
pound 1) is a strong competitive inhibitor of this enzyme with result of the rapid decrease of the diffusion coefficients of the pro-
an enhanced binding affinity relative to the natural substrate glu- tein-conjugate complexes.14,18 The shift in the oxidation potential
tathione. This conjugate exhibits an affinity for GST P1-1 approxi- to more positive values is consistent with the location of the ferro-
mately two orders of magnitude higher than that obtained for 2 cene moiety within the hydrophobic H-site of the protein. As a re-
with Schistosoma japonicum GST.19 These results also indicate that sult, the Fc unit is less exposed to the solvent and the ferrocenium
inhibitory capacity of 1 is similar to that obtained for ethacrynic ion that results from its oxidation is less stabilized by solvation. It
acid-glutathione conjugate (EA–SG), a great GST inhibitor,31 whose has been reported that the oxidation potential of some ferrocene-
EA moiety also binds strongly to H-site of GST P1-1.29 carbohydrate conjugates increases in the presence of b-cyclodex-
We have studied the electrochemical properties of ferrocene trin due to the inclusion of the ferrocene unit inside the hydropho-
glutathione conjugates 1 and 2 by differential pulse voltammetry bic cavity of the b-cyclodextrin.15
 M. C. Martos-Maldonado et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7256–7260
Figure 3. (A) Differential pulse voltammograms of compound 1 (normal line) and
compound 2 (dashed line) at 50 lM in the presence of increasing amounts of GST
P1-1 (0-90 lM) in 10 mM phosphate buffer (pH 7.2) with 20 mM NaCl as
supporting electrolyte. B) Intensity sensitivity parameters (PS,I; (I0 I)/I0) of
compounds 1 (grey) and 2 (black) at 50 lM in the presence of different
concentrations of GST P1-1 (10, 50 and 90 lM). C) Potential sensitivity parameters
(PS,E; (E0 E)/E0) of compounds 1 (grey) and 2 (black) at 50 lM in the presence of
different concentrations of GST P1-1 (50 and 90 lM).
This behavior of conjugates 1 and 2 in the presence of GST P1-1
is different to that reported for other ferrocene conjugates used as
electrochemical probes for sensing proteins in which the protein
only induces a decrease in the peak current, meaning that despite
the complexation the ferrocene moiety remains relatively exposed
to the solvent and therefore the difference between the oxidation
potentials of the ferrocene moieties of the free and protein-com-
plexed is negligible.14,19 These results obtained for the protein
GST P1-1 are different to those reported for SjGST18 and the differ-
ent behavior of such proteins towards ferrocene conjugates can be
explained by the different architecture and hydrophobic nature of
the H-site in both GSTs. This different behavior could be used as a
distinctive feature between both transferases.
To study the sensing abilities of the conjugates we defined two
sensitivity parameters to evaluate the extent of the variation of
7259
both the peak current and the oxidation potential induced by the
interaction with the protein. The peak current-based sensitivity
parameter (PS,I) is defined as (I0 I)/I0 where I0 and I denote the
peak current for the oxidation of the ferrocene moiety in the ab-
sence and the presence of GST P1-1, respectively. As can been seen
in Figure 3, based on the peak current variation, compound 1 is
slightly more sensitive than 2 at any concentration of GST P1-1.
We also defined an oxidation potential-based sensitivity parameter
(PS,E) as (E E0)/E0 where E and E0 denote the oxidation potential in
the presence and the absence of GST P1-1, respectively. Unlike the
peak current values, the E values are independent on the diffusion
coefficient. This parameter is particularly visible when the protein/
conjugate ratio is above 1. In those cases the predominant electro-
active species is the complexed form, and therefore the shift in the
oxidation potential is clearly observed. Thus, at a 1:5 ratio (10 lM
protein–50 lM conjugate), although conjugates 1 and 2 are sensi-
tive to the presence of the protein, according to PS,I, no shift in the
oxidation potential is observed (PS,E  0). Conjugates 1 and 2 are
quasi equally sensitive to the presence of the protein at 50 lM
GST P1-1. However, at 90 lM GST P1-1 compound 1 exhibited a
PS,E value twofold higher than that for compound 2 (Fig. 3).
In summary, we have examined the inhibitory potency and the
sensor properties of two ferrocene conjugates toward dimeric hu-
man glutathione S-transferase. The presence of a different spacer
arm between the ferrocene and glutathione moieties modifies
greatly the inhibitory potency to this enzyme. Thermodynamic
parameters and activity assays indicate a higher affinity with the
increase of the length of the spacer arm of the inhibitor as a conse-
quence of an increase in the entropy change due to binding. Vol-
tammetric measurements for the binding of these ferrocene
derivatives showed a strong decrease of the peak current intensity
and an increase of the oxidation potential, with the increase of the
protein concentration. Therefore, these compounds can be used as
dual electrochemical sensors for GST P1-1. The change in the oxi-
dation potential, which is only observable at higher protein/ferro-
cene conjugate ratio than one, could be used both as criteria to
discriminate between GST classes and for the detection of high lev-
els of GST P1-1.
Acknowledgments
We acknowledge the Andalusian Government (Grant CVI-6028)
and Spanish Ministry of Science and Innovation and the EU Euro-
pean Regional Development Fund (Grant CTQ2010-17848). The
Spanish Ministry of Education is also acknowledged for a scholar-
ship (M.C.M.-M.).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.09.022.
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21. The conjugation reaction (GSH–CDNB) was continuously recorded
spectrophotometrically during 2 min at 340 nm and 25 °C (e = 9600 M 1 cm 1)
in a Cary BIO 50 spectrophotometer (Varian). All GST activity assays were
performed in conditions of linearity respect to incubation time and protein
concentration. Initial rate of product formed was determined from slope of the
increase in absorbance per minute at 340 nm. These rates measured as
absorbance/time were converted in concentration/time using the indicated
molar absorptivity.
22. The human glutathione S-transferase protein (Acc. no. NP_000843.1) was
expressed in Escherichia coli, JM109 strain, using a synthetic codon optimized
gene (GeneArt, Invitrogen) cloned in the pQE60 (QIAGEN) expression vector.
Recombinant GST P1-1 enzyme was purified by affinity chromatography on
immobilized GSH. After affinity purification, the enzyme was homogeneous as
judged by SDS–PAGE.
23. Varying concentrations of 1 and 2 (0–120 lM), pre-dissolved in 0.1 M
potassium phosphate buffer (pH 7.0), were added to a reaction mixture of
1 mL (final volume) of 0.1 M potassium phosphate buffer (pH 6.5), containing
1 mM GSH and about 1 lg of GST P1-1 at 25 °C.
24. The apparent Km and Vmax values of GST P1-1 were also determined in the
presence of 1 and 2. The reaction was initiated upon addition of the substrate
1-chloro-2,4-dinitrobenzene (CDNB, final concentration 1 mM). Kinetic data
were collected by varying GSH (0.04–2 mM) at a fixed CDNB concentration
(1 mM), with a fixed inhibitors concentration.
25. Copeland, R. A. Enzymes: A Practical Introduction to Structure, Mechanism, and
Data Analysis; Wiley-VCH: New York, 2000. pp. 273–277.
26. Calorimetric experiments were conducted in an ultrasensitivity VP-ITC
instrument (MicroCal Inc., Northampton, MA). Titrations were routinely
performed in 20 mM sodium phosphate, 5 mM NaCl, 0.1 mM EDTA, pH 7.0.
Changes in the standard free energy DG0 and entropy DS0 were determined as
DG0 = RTlnK and TDS0 = DH DG0 (assuming that DH = DH0).
27. Ortiz-Salmerón, E.; Nuccetelli, M.; Oakley, A. J.; Parker, M. W.; Lo Bello, M.;
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W.; García-Fuentes, L. Protein Sci. 2009, 18, 2454.
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P.; Lo Bello, M.; Parker, M. W.; García-Fuentes, L. J. Mol. Recognit. 2011, 24, 220.
30. The binding of 1 and 2 conjugates to GST P1-1 was followed by intrinsic
fluorescence measurements a 339 nm with a PTI QuantaMaster (QM4-CW)
spectrofluorometer equipped with a Peltier device and associated with a
Biologic SFM/20 titration accessory. The excitation wavelength was 278 nm
and the emission slit widths used for excitation and emission were 2 nm. The
fluorescence measurements were corrected for dilution and inner filter.
Samples containing 2–4 lM of GST in 2 mL of 20 mM sodium phosphate
buffer (containing 5 mM NaCl, 0.1 mM EDTA) at pH 7.0 were added to a 3 mL
quartz fluorescence cell and the fluorescence intensity measured at 25 °C. A
suitable amount of the conjugated dissolved in the same buffer was then added
to each sample and the fluorescence intensity measured after mixing.
31. Nieslanik, B. S.; Ibarra, C.; Atkins, W. M. Biochemistry 2001, 40, 3536.
32. Electrochemical measurements were carried out in sonicated, nitrogen-purged
H2O (MilliQ 18.2 MXcm) solution with a microAutolab type III. The electrodes
were carefully cleaned before each experiment. The glassy carbon disk working
electrode (Ø 2 mm, effective area 0.038 ± 0.006 cm2) was immersed in a 0.1 M
HNO3 solution for 5 min and polished with a basic Al2O3–water slurry The
platinum sheet counter electrode (6 ± 4 mm, effective area 0.410  0.003 cm2)
was immersed in a 50% v/v H2SO4 solution for 5 min. Both electrodes were then
sonicated in a 1:1:1 H2O–MeOH–CH3CN mixture for 5 min prior to use. A Ag/
AgCl (3 M KCl) electrode was used as a reference. Differential pulse
voltammetric (DPV) experiments were carried out in 10 mM phosphate
buffer (pH 7.2) with 20 mM NaCl. Solutions of each conjugate (50 lM) and
increasing amounts of GST P1-1 were prepared in this buffer and shaken for
10 min at room temperature. Before each experiment, nitrogen was bubbled
for 3 min. A DPV experiment was then measured between 200 mV and
+600 mV with a scan rate of 5 mV s 1, a step potential of 20 mV, a modulation
amplitude of 50 mV, a modulation time of 0.05 s and an interval time of 2 s.
33. Bard, A. J.; Faulkner, L. R. Electrochemical Methods. Fundamentals and
Applications, 2nd ed.; John Wiley & Sons: New York, 2001. pp. 286–293.