908
D. L. Potapenko et al.
of rate constants of thiol–disulfide exchange of BSSG
with thiols. Indeed, it was found that addition of 20 mM
of GSSG to 5 mM of BSH does not significantly change
the shape of NMR 19F spectrum during half an hour.
Nevertheless, the appearance of a low signal of BSSG (5%
from added BSH, data not shown) was observed after five
hours of incubation. Therefore, the value of rate constant
of the reverse reaction (2) can be estimated to be in the
range 1.4 ð 10ꢀ3 –1.4 ð 10ꢀ4 Mꢀ1 cꢀ1. Taking into account the
obtained estimations of the equilibrium constants K1 and K2,
the value of rate constant of the direct reaction (2) should
be about 1.4–14 Mꢀ1 cꢀ1. This latter value is in the range of
literature data for the rate constants for the thiol–disulfide
exchange reactions.7 On the basis of the obtained data, it
should be expected that the NMR lines of BSSB broaden
after addition of GSH, due to exchange reaction (8) with
BSH formed. The calculations of the dependence of the 19F
NMR linewidth of BSSB on GSH concentration using a rate
constant of the reaction (8) k D 63 Mꢀ1cꢀ1 are in excellent
agreement with experimental data (Fig. 7).
Moreover, using the obtained value for the rate constant
of the exchange reaction (8) and Eqn (10) it is easy to
show that linewidth of the BSSB signals under a BSH
concentration of 1.5 mM should be about 100 Hz. This value
is 10 times higher than the linewidth of BSSB before addition
of BSH and twice the width of the whole multiplet signal
of BSSB (¾50 Hz, Fig. 3). These estimations explain the
experimentally observed linear dependence of the width
of whole multiplet signal of BSSB on BSH concentration
when [BSH] >1.5 mM (Fig. 5).
Initial broadening of NMR 19F spectral lines of BSSB
(LW0BSSB) and BSH (LW0BSH) can be explained by the presence
of BSH and BSSB, respectively, in their solutions. The
presence of BSSB in BSH solutions is easily explained by
oxidation of BSH by oxygen in the environment. Slight
concentrations of BSH in the solution of BSSB can originate
from hydrolysis of the latter.
The method also allows the measurement of thiol groups
within proteins or other macromolecules. However, an
interpretation of the NMR data in this case may be more
complex and less informative as was demonstrated for
the case of hemoglobin and albumin. Bovine hemoglobin
is a quaternary-structure protein containing two pairs
of polypeptide chains and four groups of heme. This
structure contains two cysteine residues easily available
for thiol–disulfide exchange.2–4 Therefore, at least three
different products can be formed in the reaction of Hem
with BSSB. However, in the case of complete modification
of the SH groups of hemoglobin, the formation of a single
double disulfide is expected.
the formation of one additional product in the reaction of
ASH with BSSB is expected:
BSSB C ASH ꢀ! BSSA C BSH
ꢃ13ꢂ
The formation of the disulfide of albumin as well as the
disulfide of hemoglobin is sterically hindered because of the
high molecular weights of the reagents.
NMR spectral lines of BSSA should overlap with broad
lines of BSSB (Fig. 6a). Therefore the expected ratio of the
integrated intensities of experimental NMR 19F lines of BSSB
and BSH for addition of 0.67 mM of ASH to 5 mM BSSB
solution under complete modification of albumin (13) should
be (2 ð ꢃ5 ꢀ 0.67ꢂ C 0.67ꢂ/0.67 D 13.9 which is in a good
agreement with experimentally obtained value, 14.4 š 1.5
(Fig. 6a).
From the number of available SH groups, the expected
line broadening of 19F spectral lines of BSSB due to the
exchange reaction (8) after addition of ASH should be
comparable with that observed after addition of GSH and
about two times higher after addition of Hem. However, as
is seen from Fig. 7, the real slopes in these cases are seven
and four times larger, respectively. Moreover, the linewidth
of formed BSH at concentrations of BSSB larger than 4 mM
after addition of ASH to BSSB is equal to 75 Hz instead of
expected 281 Hz (Eqn 11). These facts cannot be explained
by an intermediate exchange reaction model and are the
subjects of ongoing investigation.
The application of the approach for thiols detection in
rat blood shows good agreement with literature data, and,
therefore, demonstrates the applicability of the method for
the determination of thiols ex vivo.
CONCLUSIONS
The technique developed for thiol determination described
here is based on thiol–disulfide exchange between the
newly synthesized disulfide compound, BSSB, and thiols.
Sensitivity of linewidth of BSSB to the concentration of
SH compound allows determination of 50 µmol lꢀ1 of
thiol (corresponding to a linewidth broadening of 3 Hz)
which is slightly lower than the sensitivity of EPR-based
technique using biradical disulfides.10,11 However, the 19F
NMR spectrum allows for an unambiguous conclusion
about the structure of the presented thiol (Fig. 8a). Stability
of the formed diamagnetic compounds in the reducing
cellular environment provides a significant advantage of
NMR approach over EPR detection.16 The NMR technique
also can be performed in nontransparent samples, which
is not possible using standard optical methods.6–9 The
rate constants for the thiol–disulfide exchange reactions
of the BSSB with thiols studied are too high for the
kinetics to be detected. Therefore an application of an excess
of BSSB over the thiols is required for thiols detection
using measurement of the equilibrium, BSSB/BSH. This
significantly limits the number of biological applications
of the proposed technique using this particular fluorinated
disulfide probe. Measurement of the line broadening of
BSSB for the thiol detection is an alternative approach that
Hem(SH)2 C 2BSSB ꢀ! Hem(SSB)2 C 2BSH
ꢃ12ꢂ
NMR 19F lines of this double disulfide should overlap
with broad lines of BSSB (Fig. 6b). While the signal at
18.00 ppm can be attributed to one of the NMR lines of
the product, the exact analysis of the spectrum presented in
Fig. 6b is complicated because of line broadening.
Albumin, ASH, is a protein with molecular weight about
65 kDa, which contains one available SH group.5 Therefore,
Copyright 2005 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2005; 43: 902–909