Analytical Chemistry Chemical Cytometry Quantitates Superoxide

plasma samples were analyzed using the teicoplanin FPIA kit
following the instruction of the manufacturer.
RESULT AND DISCUSSION
Design and Synthesis of Peptide Probes. We used aminereactive
fluorophores to label the acetylated peptide Ac-L-Lys-D-
Ala-D-Ala-OH. We chose this peptide for two reasons. First, L-lys
is naturally occurring in the peptidoglycan precursor peptide L-Ala-
D-Glu-L-Lys-D-Ala-D-Ala. We expected the incorporation of the L-lys
would not affect the interaction between the peptide and the
antibiotics. Second, the side chain of Lys contains a primary amine
group, which is distant from the binding site judged by the crystal
structure of the vancomycin and diacetyl-L-Lys-D-Ala-D-Ala complex.
34 As expected, we found that the binding affinities between
the labeled peptide and antibiotics were similar to the values
reported for the binding between the free peptide and antibiotics,
indicating that the modification did not affect their interaction (see
below).
Effect of Fluorophores. The fluorescence polarization signal
is determined by the Perrin Equation. 35
( 1 1
-
P 3) ) ( 1
-
P0 1 kT
1 + 3)( ηV τ)
Where τ is the fluorescence lifetime of the fluorophore, P0 is the
limiting polarization, k is the Boltzmann constant, T is the
absolute temperature, η is the viscosity, and V is the molecular
volume (molecular weight). The fluorescence polarization is
negatively correlated with the lifetime of the fluorophores and
positively correlated with the molecular volume (molecular
weight). In order to maximize the observed change of polarization
upon antibiotic binding, we tested two fluorophores with
different lifetimes, FITC (4 ns lifetime) and AF680 (1 ns
lifetime) (Figure 1). We found that AF680-peptide yielded a larger
change of signal upon drug binding and, thus, exhibited a better
signal-to-noise ratio.
Titration Curves of Glycopeptide Antibiotics in Phosphate
Buffer. The binding between the antibiotics and peptide probes
was monitored following the increase of fluorescence polarization
of the samples upon addition of increasing concentrations of the
antibiotics (Figure 1). The dissociation constants (Kd) between
the drugs and peptide probes were estimated to be around 1.1,
0.2, and 1.8 µM, respectively, for vancomycin, teicoplanin, and
telavancin (Table 1). The identity of the fluorophores did not have
a drastic effect on the binding affinity, suggesting they were not
directly involved in the interaction. The determined affinities are
similar to the values reported for the bindings between these
antibiotics and diacetyl-L-Lys-D-Ala-D-Ala (∼1 µM for vancomycin,
and ∼0.6 µM for teicoplanin). 36-39 Compared with the FITC-
(34) Nitanai, Y.; Kikuchi, T.; Kakoi, K.; Hanmaki, S.; Fujisawa, I.; Aoki, K. J.
Mol. Biol. 2009, 385, 1422–1432.
(35) Ye, B. C.; Ikebukuro, K.; Karube, I. Nucleic Acids Res. 1998, 26, 3614–
3615.
(36) Popieniek, P. H.; Pratt, R. F. Anal. Biochem. 1987, 165, 108–113.
(37) Azad, M.; Hernandez, L.; Plazas, A.; Rudolph, M.; Gomez, F. A. Chromatographia
2003, 57, 339–343.
(38) Rao, J. H.; Yan, L.; Xu, B.; Whitesides, G. M. J. Am. Chem. Soc. 1999, 121,
2629–2630.
(39) van Wageningen, A. M. A.; Staroske, T.; Williams, D. H. Chem. Commun.
1998, 1171–1172.
7046 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
(2)
Figure 1. Calibration curves of FITC-peptide (diamonds) and AF680peptide
(squares) with vancomycin (A), teicoplanin (B), and telavancin
(C) in PBS. The concentration of the peptide probes were 1 µM. The
concentrations of the glycopeptide antibiotics increased from 0 to 7.7
µM during the titration. The data were fitted using a one-to-one binding
model to derive the dissociation constants listed in Table 1.
Table 1. Fitting Parameters of Peptide-Drug
Interaction
glycopeptide
antibiotics probe matrix
dissociation
constant Kd (µM)
vancomycin FITC-peptide PBS 0.91 ± 0.06
AF680-peptide PBS 1.07 ± 0.19
AF680-peptide FBS 1.81 ± 0.13
teicoplanin FITC-peptide PBS 0.14 ± 0.07
AF680-peptide PBS 0.22 ± 0.02
AF680-peptide FBS 0.87 ± 0.08
telavancin FITC-peptide PBS 3.22 ± 0.38
AF680-peptide PBS 1.81 ± 0.36
AF680-peptide FBS 5.36 ± 0.35
peptide probe, the AF680-peptide yielded a better signal-to-noise
ratio. Therefore, it was used in fluorescence polarization assays
for the rest of the study.
To directly examine if the free fluorophores (FITC or AF680)
interacted with the glycopeptide antibiotics, we compared the
change of fluorescence polarization of 1 µM FITC, AF680, FITCpeptide,
or AF680-peptide upon addition of 8 µM vancomycin,
teicoplanin, or telavancin (Figure 2). No significant change of
fluorescence polarization was observed for samples containing free
fluorophores, indicating the lack of interaction between the drugs
and the free fluorophores.
Drug Detection in Serum. To further examine the usefulness
of the current method in the detection of glycopeptide antibiotics
in clinical samples, we measured the titration curves using AF680peptide
for the detection of vancomycin, teicoplanin, and telavancin
in FBS. The potential matrix effects from serum could affect the
assay in two ways. First, the higher viscosity in serum slows down
the tumbling of the fluorophores and, thus, increases fluorescence
polarization. Second, certain species in the serum may interact
with the peptide probe or the drugs to prevent accurate detection.