Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Figure 1. (A) FL spectra of BSPOTPE in the PBS buffer containing different amounts of HSA. (B) Change in the FL intensity at 475 nm with
HSA concentration; I0 ) FL intensity in the absence of HSA. Inset: linear region of the binding isotherm of BSPOTPE to HSA. [BSPOTPE] )
5 µM; λex ) 350 nm.
differential scanning calorimetry (DSC), and molecular modeling
helped draw a mechanistic picture on the protein unfolding
process.
MATERIALS AND METHODS
General Information. BSPOTPE was prepared according to
our previously published procedures. 21b HSA, GndHCl, and other
biomolecules were all purchased from Sigma and used as received.
Phosphate buffered saline (PBS) with pH of 7.0 was purchased
from Merck. Water was purified by a Millipore filtration system.
All the experiments were performed at room temperature unless
otherwise specified.
UV spectra were measured on a Milton Roy Spectronic 3000
Array spectrophotometer, and FL spectra were recorded on a
Perkin-Elmer LS 55 spectrofluorometer with a Xenon discharge
lamp excitation. CD spectra were recorded on a Jasco J-810
spectropolarimeter in a 1 mm quartz cuvette using a step
resolution of 0.2 nm, a scan speed of 100 nm/min, a sensitivity of
0.1°, and a response time of 0.5 s. Each spectrum is the average
of three scans. Details about the artificial urine preparation,
cytotoxicity assay, FRET study, DSC and pH-dependent FL
measurements, and computation modeling are given in the
Supporting Information.
Sample Preparation. Stock solutions of BSPOTPE and HSA
with a concentration of 1.0 mM were prepared by dissolving
appropriate amounts of the luminogen and protein in the PBS
buffer. The final concentration of HSA in PBS was double checked
by measuring its absorbance at 279 nm. In the HSA unfolding
study, HSA (0.4 µM) in PBS was incubated in the presence of
different amounts of GndHCl (0.2-7.0 M) at 25 °C for 30 min.
Afterward, BSPOTPE was added to the mixtures and incubated
for another 30 min before spectral measurements. FL spectra were
recorded in the wavelength range of 370-670 nm using 350 nm
as the excitation wavelength. For the intrinsic tryptophan fluorescence
study, FL spectra were collected from 310 to 570 nm
using 290 nm as the excitation source. In the refolding experiment,
0.2 mM HSA was incubated in 8 M GndHCl for 24 h at 25 °C to
ensure that all the proteins were fully unfolded. The denatured
sample was then diluted with PBS until the final concentrations
of HSA and GndHCl were equal to 0.4 µM and less than 0.1 M,
respectively. Fraction of refolded proteins (F r) was calculated
from the following equation:
F r ) 1 - I N - I R
I N - I D
where IN and ID are the FL intensities of native and denatured
HSA-BSPOTPE complexes, respectively, and IR is the FL
intensity of BSPOTPE recovered from the refolded HSA.
Electrophoresis Assay. A poly(acrylamide) gel electrophoresis
(PAGE) experiment was performed on a Hoefer miniVE system
under nondenaturing conditions using 5% stacking and 12%
resolving native poly(acrylamide) gel at 100 V for 3hat4°C.
After electrophoresis, the gel was soaked in an aqueous solution
of BSPOTPE (1 mg in 100 mL ddH2O) at room temperature for
5 min. Alternatively, the gel was stained with a Coomassie
Brilliant Blue (CBB) solution (0.1% w/v Coomassie Blue R250
in an aqueous mixture containing 10% methanol and 7% acetic
acid) for6htoovernight on a rotary shaker with gentle mixing,
followed by destaining in an aqueous solution containing 10%
methanol and 7% acetic acid for 1 to 2 h until the background
of the gel became transparent. An AlphadigiDoc system with
a DE-500 MultiImage II light cabinet and an ML-26 UV
transilluminator (Alpha Innotech) was used for data collection
and analysis.
RESULTS AND DISCUSSION
Protein Quantitation in Solution. BSPOTPE is soluble in
water but insoluble in common organic solvents, such as acetonitrile,
THF, and chloroform. Its FL quantum yield (ΦF) is
increased from 0.37% in water to 17.5% in acetonitrile, where
the luminogen molecules aggregate. The BSPOTPE solution
in PBS is feebly luminescent at 390 nm in the absence of HSA
(Figure 1A). When a small amount of HSA is added, the
BSPOTPE solution becomes luminescent. Its FL intensity at 475
Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
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