2
Y. Byun et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 221 (2019) 117151
containing fluorescent eosin-labeled GSH (E-GSH) the upper axial li-
gand to the cobalt (Scheme 1). The fluorescence of E-GSH was strongly
quenched in E-GSCbl. The E-GSH ligand of E-GSCbl was replaced specif-
ically by cyanide, showing recovery of the E-GSH fluorescent, which en-
abled fluorescence turn-on detection of cyanide at nanomolar
concentrations (Scheme 2). In addition, E-GSH exhibited strong lumi-
nescence under UV-light, which was also quenched in E-GSCbl, and
this allowed straightforward naked-eye detection of cyanide. These re-
sults demonstrate that profluorescent E-GSCbl is a highly sensitive
chemosensor that is able to detect nanomolar concentrations of
cyanide.
2.3. HPLC analysis
Cobalamins were analyzed by HPLC as previously described [21]. A
reaction mixture contained 50 μM E-GSCbl and 200 μM KCN in 50 mM
Tris/HCl pH 7.5. After 2 h incubation in the dark at room temperature,
the reaction mixture was loaded on an ODS-3 V C18 reversed phase col-
umn (250 × 4.6 mm, 5 μm, GL Sciences) equilibrated with solvent A
2
(0.1% (v/v) trifluoroacetic acid/H O). The column was then washed
with solvent A for 5 min and eluted with a linear gradient from 0 to
40% solvent B (0.1% (v/v) trifluoroacetic acid/acetonitrile) over 40 min
at a flow rate of 1 mL min− , eluent was monitored at 255 nm. Standard
1
2
cobalamins OH Cbl, CNCbl and E-GSCbl were eluted at retention times
of 17.9 min, 21.2 min and 23.0 min, respectively. The retention time of
the cobalamin product obtained in the reaction of E-GSCbl with KCN
was compared with the retention times of standard cobalamins.
E-GSH released from E-GSCbl by cyanide replacement was identified
by HPLC analysis with some modifications [19,21]. Briefly, reaction mix-
tures contained 50 μM E-GSCbl and 200 μM KCN (5200 μg L− ) in 50 mM
Tris/HCl pH 7.5. After 2 h incubation in the dark at room temperature,
reaction mixtures amino groups of glutathione were derivatised with
2
. Materials and methods
2
.1. Materials
Chemicals of analytical grade were purchased from Sigma Aldrich,
1
unless otherwise indicated. Potassium cyanide (KCN) solution was
freshly prepared in 20 mM NaOH and neutralized before use and a cya-
nide standard from Sigma was used to control the quality of determina-
2
,3-dinitrofluorobenzene flowing the reaction of free thiols with
monoiodoacetic acid and injected on a on a Bondclone NH column
300 mm × 3.9 mm, 10 μm, Phenomenex) equilibrated with solvent C
80% (v/v) methanol/water). The column was washed with 60% solvent
−
−
−
−
−
2−
2−
tion methods. Stock solutions (500 mM) of Cl , F , Br , I , SCN , S , SO
3
,
2
−
, HSO−
, NO−
, HCO−
3−
2−
2
S
2
O
3
3
3
3
, PO
4
or SO
4
were prepared by dissolving
(
(
proper amounts of sodium salts in distilled and deionized water.
D (a mixture of 400 mL of solvent A with a 100 mL solution of 272 g so-
dium acetate trihydrate, 122 mL water and 373 mL glacial acetic acid)
for 5 min and eluted with a gradient of 60–100% solvent D over
40 min at a flow rate of 1 mL min− with eluent monitoring at
255 nm. E-GSCbl and E-GSH were eluted at retention times of
11.6 min and 13.5 min, respectively. The retention time of E-GSH prod-
uct generated in the reaction of E-GSCbl with KCN was compared with
the retention time of the E-GSH standard.
2
.2. Synthesis of eosin-labeled glutathione and glutathionylcobalamin
1
Eosin-labeled glutathione disulfide (Di-E-GSSG) was prepared, as
described previously [17], and concentrations of Di-E-GSSG were deter-
mined by using the extinction coefficient of ε523 nm = 112 mM− cm
1
−1
.
Eosin-labeled glutathione (E-GSH) was prepared by reducing Di-E-
GSSG with 40-fold molar excess dithiothreitol (DTT). E-GSH was precip-
itated by adding 10-volume of ice-chilled acetonitrile (CAN) and
centrifuging to remove excess DTT. E-GSH concentrations were deter-
mined by using an extinction coefficient of ε519 nm = 88 mM− cm
1
−1
2.4. Spectrophotometric assays
[18].
Eosin-labeled glutathionylcobalamin (E-GSCbl) was synthesized by
following a patented method (US patent number 7,030,105) with
some modifications, as previously described [19]. Briefly,
Spectrophotometric assays contained 10 μM E-GSCbl in 50 mM Tris/
HCl pH 9.0 and initiated by the addition of the indicated concentrations
of KCN. Reactions were followed under dark conditions for 2 h at room
temperature by recording absorption spectra using a Cary 100 UV–Vis
spectrophotometer (Varian). CNCbl generation was calculated by plot-
ting A361 nm versus KCN concentrations by simple linear regression
aquacobalamin (OH
2
Cbl) was mixed with E-GSH at a molar ratio of
1
:1.5 and the generation of E-GSCbl was followed by recording changes
in the absorption spectrum. After incubation for 2 h in the dark at room
temperature, E-GSCbl was precipitated by adding 10-volume of ice-
chilled ACN and excess E-GSH was removed by centrifugation. E-
GSCbl precipitates were dissolved in appropriate buffer and E-GSCbl
concentrations were determined by using the extinction coefficient of
analysis (Δε361 nm = 14.2 mM− cm ) [14]. The detection limit of
the spectrophotometric assay was determined according to IUPAC rec-
ommendation [22]: lower limit of detection = 3SD/s, SD is the standard
deviation of the blank measurements (n ≥ 7), s is the slope of the titra-
tion curve.
1
−1
ε372 nm = 14 mM− cm [20].
1
−1
Scheme 1. Chemical structures of eosin-labeled glutathionylcobalamin and glutathione. R is the upper axial ligand and DMB (5,6-dimethylbenzimidazole) is the lower axial ligand to the
cobalt in cobalamin (left). Eosin-labeled glutathionylcobalamin (E-GSCbl) contains eosin-labeled glutathione (E-GSH) is the upper axial ligand (right).