Journal of the American Chemical Society
ARTICLE
heme required for optimal cellular function, as well as catalyze 1-
and 2-electron oxidative reactions that are detrimental to the cell.
Given the ease of oxidation of indole and its derivatives by
nucleoheme complexes, it is also interesting to note that oxida-
tion of externally administered indoleacetic acid (IAA) by
peroxidases has shown promise as an anticancer therapeutic stra-
tegy.59 It will be intriguing to see if nucleoheme complexes can be
brought to participate in such a strategy and perhaps offer
advantages over the use of potentially immunogenic protein
peroxidases.
0.05% Triton X-100, and 1% DMF), at 21 °C, containing 1 mM H2O2, 1
mM indole, 10 μM Fe(III) heme, and 25 μM DNA/RNA. The lower
DMF content of this buffer, relative to that used for thioanisole oxida-
tion, is in response to the higher aqeuous solubility of indole. At specified
times, 95 μL of each reaction mixture was supplemented with 5 μL of 1
mM benzophenone (internal standard) and then immediately placed at
-80 °C to stop the reactions. Samples were later thawed and analyzed
using HPLC. Details of the HPLC runs are given in the Supporting
Information.
Indigo Cuvette Image Protocol. Each reaction was carried out
on a 500 μL scale. Reactions consisted of indole (2 mM), DNA (25 μM),
and Fe(III) heme (10 μM) in I-oxidation buffer. A 5 μL volume of
hydrogen peroxide (100 mM) was added to specific cuvettes, and not to
other cuvettes, and the contents were mixed. After approximately 5 min,
the blue color reached saturation; the cuvettes were then set up on a
white light box for photography. The brightness and contrast for the
images were later optimized.
’ EXPERIMENTAL SECTION
Materials. All DNA was purchased from Integrated DNA Tech-
nologies, Inc. All RNA was purchased from University Core DNA
Services (University of Calgary). The sequences of all DNAs and RNAs
are given in Table 1. All nucleic acids were purified by preparative gel
electrophoresis, eluted, ethanol precipitated, and then stored dissolved
in TE buffer [10 mM Tris, pH 7.5, and 0.1 mM ethylenediaminete-
traacetate (EDTA)]. All chemicals were purchased from Sigma-Aldrich,
unless specified otherwise. Fe(III) heme (hemin) was purchased from
Porphyrin Products (Logan, UT). 18O-Hydrogen peroxide was pur-
chased from Icon Isotopes (Summit, NJ).
UV-Vis Spectroscopy of Fe(III) Heme and Nucleoheme
Complexes. A 1 mL solution containing 1 μM PS2.M or SS18 and 0.5
μM Fe(III) heme in spectroscopy buffer [50 mM MES (2-(N-morpho-
lino)ethanesulfonic acid), pH 6.2, 100 mM Tris-acetate, 20 mM
potassium acetate, 0.05% Triton X-100, 1% DMSO] was incubated
for 30 min at 21 °C to permit DNA-heme interactions, where possible.
Spectra taken in oxidation buffer and in I-oxidation buffer (see below)
give the same results. The UV-vis spectra of PS2.M with Fe(III) heme,
SS18 with Fe(III) heme, and Fe(III) heme alone were obtained using a
Cary 300 Bio UV-vis spectrophotomer. Any background from buffer
alone was subtracted from the sample spectra.
Styrene Oxidation. See the Supporting Information.
Heme Docking upon the Bcl-2 DNA G-Quadruplex. See the
Supporting Information.
’ ASSOCIATED CONTENT
S
Supporting Information. Details of experimental proto-
b
cols and data on the enantioselectivity and H2O2 utilization
properties of nucleoheme complexes for oxygen transfer reac-
tions. This material is available free of charge via the Internet at
’ AUTHOR INFORMATION
Corresponding Author
Time Course Measurements on Thioanisole Sulfoxida-
tion. A 32 μL volume of a 100 μM DNA stock in TE buffer and 10
μL of 100 μM Fe(III) heme in DMF were added to 500 μL of a 2ꢀ
buffer (80 mM HEPES-NH4OH, pH 8.0, 40 mM KCl, 0.1% Triton
X-100, and 2% DMF) in a 1.5 mL glass vial. ddH2O was added to make
the volume 980 μL. The solution was incubated for 5 min at 21 °C to
allow for DNA-heme interactions. A 10 μL volume of 20 mM
thioanisole in DMF was added to the solution, and the resulting solution
was vortexed to mix. Prior to the start of the oxidation reaction, a 99 μL
aliquot was set aside for time 0 (and was treated as described below).
The reaction was initiated by the addition of 9 μL of 100 mM H2O2. The
resulting 900 μL volume containing 0.2 mM thioanisole, 1 μM Fe(III)
heme, 3 μM DNA, and 1 mM H2O2 in oxidation buffer (40 mM
HEPES-NH4OH, pH 8.0, 20 mM KCl, 0.05% Triton X-100, 3% DMF)
was incubated at room temperature. Aliquots of 95 μL of the reaction
mixture were removed at time 0 and 15 s, 30 s, 1 min, 2 min, 5 min, and
30 min after initiation of the reaction. A 5 μL volume of 1 mM
benzophenone was added as an internal standard to each aliquot prior
to addition of 200 μL of CH2Cl2, both to quench the reaction and to
extract the contents of the aqueous reaction mixture, which was analyzed
using HPLC (see the Supporting Information).
’ ACKNOWLEDGMENT
We are grateful to Erika Plettner, Peter Unrau, and the
laboratories of Robert Young, Gerhard Gries, and Robert Britton
for their advice and for access to their equipment. We also
appreciate the help of the technical staff of the Simon Fraser
University Chemistry Department. This work was supported by
a grant to D.S. from the Natural Sciences and Engineering
Research Council of Canada (NSERC). D.S. is a fellow of the
Canadian Institute for Advanced Research (CIFAR).
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dx.doi.org/10.1021/ja108571a |J. Am. Chem. Soc. 2011, 133, 1877–1884