Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Figure 8. Details of the best binding conformations with the lowest binding free energies in clusters (A) 1 and (B) 2 (cf., Supporting Information,
Figure S6). In each case, the BSPOTPE molecule is shown in a stick representation with a semitransparent van der Waals surface and with a
color scheme of gray for carbon, blue for nitrogen, red for oxygen, and yellow for sulfur. The residues interacting with BSPOTPE are shown as
sticks (with nonbinding apolar hydrogen atoms omitted for clarity) and labeled on R carbon. Yellow dashed lines denote hydrogen bonds.
accommodate the aromatic core of BSPOTPE via cation-π
interaction. A similar binding mode is found in clusters 2 (Figure
8B). Additionally, Pro447 contacts the hydrophobic part of the
luminogen, while His440 interacts with its sulfonate group. All
the aforesaid interactions rigidify the molecular conformation of
the BSPOTPE luminogen and hamper its intramolecular rotation,
hence making it highly emissive in the binding state. The lowest
binding energies of the conformations in clusters 1 and 2 are -8.60
and -7.54 kcal/mol, corresponding to estimated inhibition constants
(K i) of 0.49 and 2.98 µM, respectively. These small Gibbs
free energies indicate that the interaction between BSPOTPE
and HSA is highly spontaneous and energetically favorable.
The detailed binding modes of the conformations from the
other clusters in the hydrophobic cavity closely resemble those
of clusters 1 and 2, although they are grouped into different
clusters. This is not unexpected because the ligand is treated as
fully flexible in the computational modeling process, and a small
rmsd tolerance is used for clustering. Thus, any difference in the
coordinates of an atom between two conformations will increase
the rmsd value, even though the atom is not important for binding
interactions.
HSA is a physiological carrier in the human body, and its
hydrophobic regions are capable of binding insoluble endogenous
compounds like fatty acids and drugs. With close scrutinization
of other proteins, such as trypsin, pepsin, and papain, one can
hardly find a hydrophobic pocket as in HSA. This may explain
why the BSPOTPE luminogen shows excellent selectivity toward
the albumin proteins over the other types of proteins.
GndHCl is known to work as a bulky ionic cosolvent. It not
only weakens hydrophobic interaction but also interferes with
association between charged solutes. 39 When HSA unfolds, a large
number of nonpolar side chains are exposed to the aqueous
medium and stabilized by GndHCl. GndHCl may also intrude into
the hydrophobic pocket of HSA and break down the spatial
architecture of the protein folds. CD spectral data indicate that
no significant alternation in the secondary structure occurs during
the first unfolding transition in the low concentration range of
GndHCl (6.0 M). No secondary
structure or strand helicity is retained in this region, as confirmed
by the CD spectral data. The luminogen molecules are fully
released from the protein, and no emission signals can be
collected. The unfolding process discussed above is summarized
in Scheme 1. Upon removal of the GndHCl denaturant by dilution,
the protein returns to its native form and the emission of
BSPOTPE is recovered, indicating that the interaction between
HSA and BSPOTPE is fully reversible.
CONCLUDING REMARKS
In summary, in this work, we developed an environmentally
stable and synthetically readily accessible FL probe for HSA
detection and quantitation. The nonluminescent BSPOTPE becomes
emissive in the presence of HSA. The AIE bioprobe shows
a linear calibration curve at [HSA] ) 0-100 nM, enabling the
protein quantitation over a wide concentration range. It enjoys a
low detection limit (down to 1 nM) and a high selectively toward
albumins. The FL bioassay is tolerant of the species in the artificial
urine and is promising for applications in real-life urinary protein
detection. Design and fabrication of simple bioassay kits for clinical
diagnostics are ongoing in our laboratories.
BSPOTPE is successfully employed as a protein staining
reagent in the PAGE analysis. It offers a simple, rapid, effective,
and economic way for visualization of protein bands in the gel
assay. Studies on the interaction between BSPOTPE and HSA