Transcript DHE and the Specific Detection of Superoxide B. Kalyanaraman, Ph.D. Department of Biophysics Free Radical Research Center Twelfth Annual Meeting of the Society for Free Radical.

DHE and the Specific
Detection of Superoxide
B. Kalyanaraman, Ph.D.
Department of Biophysics
Free Radical Research Center
Twelfth Annual Meeting of the
Society for Free Radical Biology and Medicine
Austin, Texas
November 16-20, 2005
Reaction between Hydroethidine
and Superoxide
NH 2
H2 N
Superoxide anion
H
N
CH2CH3
Hydroethidine (HE)
NH 2
H2 N
+
N
CH2CH3
Ethidium (E+)
573 nm 593 nm
Fluorescence Spectra of the Product
586 nm
Formed during Oxidation of HE by X/XO
605 nm
14
595 nm
605 nm
Intensity
12
573 nm
HE + X + XO
HE
E+
10 586 nm
8
6
4
573 nm 593 nm
595 nm
2
0
550
600
650
700
750
800
Wavelength (nm)
Incubation mixtures consisted of HE (50 μM), xanthine (1 mM), DTPA (100 μM),
−1
and XO 586
(0.05
nmU ml ) in aerated phosphate buffer (100 mM, pH 7.4). Reaction was
initiated by the addition of XO and the fluorescence spectrum recorded after 30
min. Shown for comparison is the spectrum obtained from E+ (50 μM) obtained
under identical experimental conditions.
Zhao et al. FRBM 34:1359-1368 (2003).
Does superoxide react with
hydroethidine to form ethidium?
NH 2
H2 N
Superoxide anion
H
N
CH2CH3
Hydroethidine
NH 2
H2 N
???
+
N
CH2CH3
Ethidium
Superoxide Generating Systems
1. Xanthine/Xanthine Oxidase Systems
Xanthine (1 mM) + O2
Xanthine
Oxidase (0.05U/ml)
Uric acid + O2
2. Potassium superoxide (KO2) in DMSO with crown ether
3. BH4-depleted eNOS systems
Ca2+/ CaM
FMN
FMN
HEME
FAD
NADPH
L-Arg
FAD
NADPH
HEME-O2
L-Arg
O2•–
605 nm
HE + X + XO + DNA,
0 min
200
5 min
10 min
150
15 min
E++ DNA
80
60
595 nm
40
100
20
0
50
550
600
650
700
750
Incubation mixtures consisted of HE (50 μM), xanthine
(1 mM), DTPA (100 μM), and XO (0.05 U ml−1) in
aerated phosphate buffer (100 mM, pH 7.4). Reaction
was initiated by the addition of XO and the fluorescence
spectrum recorded after 30 min. Shown for comparison
is the spectrum obtained from E+ (50 μM) obtained
under identical experimental conditions. (A) in the
presence of DNA (250 μg ml−1). (B) Incubations
contained HE (50 μM), DTPA (100 μM), and DNA (250
μg ml−1) and xanthine (1 mM) or XO (0.05 U ml−1), both
in the presence and absence of SOD (20 μg ml−1). (C)
Same as (B) but in the presence of catalase (1000 U
ml−1).
0
800
Wavelength (nm)
120
C
573 nm
HE + DNA
HE + X + DNA
HE + XO + DNA
HE + X + XO + DNA
HE + X + XO + SOD
+ DNA
100
Intensity
Intensity of product-DNA
B
80
60
40
586 nm
20
0
550
600
650
700
Wavelength (nm)
750
800
120
573 nm
593 nm
100
80
60
250
HE + X + XO + CAT
+ DNA, 0 min
5 min
10 min
15 min
E+ + DNA
40
200
+
250
593 nm
150
100
50
20
0
550
600
650
700
Wavelength (nm)
750
Intensity of E -DNA
573 nm
586 nm
Intensity of E+-DNA
100
Intensity of product-DNA
A
Fluorescence Spectra of HE/X/XO-DNA
and
EB-DNA
593
nm
573 nm
0
800
Fluorescence Spectra of HE/KO2-DNA and EB-DNA
B
200
40
30
20
HE + KO2
HE + KO2 + DNA
HE + DNA
E+
150
E++ DNA
100
586 nm
50
10
0
550
600
650
700
Wavelength (nm)
750
50
+
593 nm
581 nm
0
800
581 nm
HE + DNA
HE + KO2 (0.55 mM)
+ DNA
HE + KO2 (1.1 mM)
+ DNA
HE + KO2 (1.1 mM)
+ SOD + DNA
40
Intensity
50
Intensity of E -DNA
Intensity of product-DNA
A
30
586 nm
20
10
0
550
600
650
700
750
Wavelength (nm)
Fluorescence spectra of the product formed during oxidation of HE by potassium superoxide. (A)
Incubations consisted of HE (50 μM), DTPA (100 μM), and KO 2 in the presence and absence of
DNA (250 μg ml−1) in phosphate buffer (100 mM, pH 7.4). Superoxide solution was prepared by
adding 1 mg of KO2 in 1 ml of dry DMSO and vortexed vigorously for 10 min. The reaction was
initiated by adding 40 μl of freshly prepared KO2 in DMSO to 0.46 ml of the above buffer (final
concentration of KO2 was 1.1 mM). (B) Same as (A) but also containing SOD (20 μg ml −1). The
extent of oxidation of HE under these conditions is much less than that observed in X/XO.
800
Fluorescence spectra of HE/BH4-free eNOS-DNA
and EB-DNA
60
+ Ca2+/CaM + L-arg
+ Ca2+/CaM + L-arg
586 nm
40
+ BH4
+ Ca2+/CaM + BH4
20
0
600
650
700
800
Wavelength (nm)
573 nm
250
593 nm
5 min
10 min
15 min
20 min
25 min
30 min
E+ + DNA
60
40
20
200
150
100
50
0
550
600
650
700
750
0
800
Wavelength (nm)
60
50
Intensity
750
80
+
+ Ca2+
+ Ca2+/CaM
550
C
B
573 nm
5 min
10 min
15 min
20 min
25 min
30 min
40
586 nm
30
20
10
0
550
600
650
700
Wavelength (nm)
750
800
Intensity of E -DNA
80
Intensity of product-DNA
Intensity of product-DNA
A
(A) Incubations consisted of NADPH (0.1 mM), DTPA (100
μM), HE (50 μM), L-arginine (0.1 mM), purified eNOS (6.25
μg ml−1), and Ca2+ (0.2 mM)/CaM (20 μg ml−1) in a HEPES
buffer (50 mM, pH 7.4). Spectra were obtained 30 min after
the addition of eNOS to different incubation mixtures. There
is little or no oxidation of HE in the presence of BH4 (10 μM).
(B) Time-dependent changes in fluorescence of the product
formed from an incubation mixture containing NADPH (0.1
mM), HE (50 μM), DTPA (100 μM), Ca2+ (0.2 mM)/CaM (20
μg ml−1), and purified eNOS (6.25 μg ml−1) in a HEPES
buffer (50 mM, pH 7.4). (C) Same as (B) except in the
presence of SOD (20 μg ml−1).
Intensity
Fluorescence spectra of the product
of HE reaction with O2•– and EB-DNA
180
160
140
120
100
80
60
40
20
0
567
567
nm
550
593n
nm
593 nm
m
Product + DNA
E+ + DNA
600 650 700 750
Wavelength (nm)
800
Fluorescence spectra of the purified product formed from oxidation of
HE by X/XO and E+ in the presence of DNA. Incubations contained
50 μM of the pure product, 50 μM E+ and DNA (250 μg ml−1) in
phosphate buffer (100 mM, pH 7.4).
HPLC Analysis
A
HE 50 M
B
HE 50 M, X 1 mM,
XO 0.05 U/ml
C
EB 50 M
D
HE 50 M, X 1 mM, XO
0.05 U/ml + EB 50 M
10
15
20
Time (min)
25
30 25
26
Time (min)
27
Fluorescence EX 510 nm, EM 595 nm
HPLC chromatograms of HE, E+, and the product formed from oxidation of HE by the X/XO system. (A)
Incubations contained xanthine (1 mM), HE (50 μM), and DTPA (100 μM). (B) Same as above but in the
presence of XO (0.05 U ml−1). HPLC traces in (A) and (B) were obtained 30 min after starting the
incubation. (C) HPLC trace of authentic E+ (50 μM) in phosphate buffer (100 mM, pH 7.4). (D) Same
incubation conditions as in (B) but spiked with authentic E+ (50 μM). Right trace (A–D), HPLC
chromatograms recorded on an expanded scale. Fluorescence detection at 510 nm (excitation) and
595 nm (emission) was used to monitor HE, E+, and the oxidation product of HE.
Mass Spectrometry
Relative Abundance
A
100
80
60
330.1
Product of
HE and O2•–
40
331.1
20
0
280
B
300
Relative Abundance
100
80
60
320
m/z
340
360
340
360
314.2
Ethidium
bromide
315.2
40
20
0
280
300
320
m/z
HPLC-mass spectrometry.
(A) The mass spectrum of
new product taken from the
LC peak at retention time of
34.30 min; incubations
contained xanthine (1 mM),
HE (50 μM), and DTPA
(100 μM) and XO (0.05 U
ml−1) in phosphate buffer
(100 mM, pH 7.4). (B) The
mass spectrum of E+ taken
from the LC peak at
retention time of 33.97 min;
incubations contained E+
(50 μM) in phosphate buffer
(100 mM, pH 7.4).
Summary
1. Hydroethidine reacts with superoxide to form a
fluorescent compound, which is different from
ethidium bromide. This compound is a specific
product for hydroethidine reaction with superoxide.
The fluorescence of the new compound is also be
enhanced by DNA.
2. Other oxidants (peroxynitrite, HOCl, H2O2, hydroxyl
radical and peroxy radical) do not react with HE to
form this particular product.
3. The new compound has a different retention time and
molecular weight compared to ethidium bromide.
4. The fluorescence properties of the new compound are
different from that of ethidium bromide.
Conclusion
.
HE + O
2
.
HE + O
2
E+
Characteristic
fluorescent product
that binds to DNA
Determination of the Structure of the
HE/O2∙ Product
301.2
100
of 330
Relative Abundance
MS2
MH+ -C2H4
-NH3
C2H5• m/z 330
m/z 285
m/z 302
m/z 301
50
302.2
-H2
-H•
m/z 300
75
0
NMR
285.0
25
224.1 257.2
100
200
m/z
330.1
313.1
300
400
H4
H1
H10
H9
H7
H9
NH
A
H2N
CH2
H7
9
8
7 ppm 6
5
Zhao et al. PNAS 102:5727-32 (2005)
4
H1
H10
OH
B

N
NH2
H4
CH2CH3
Structure of the HE/O2∙
Reaction Product
9
H2N
1
10
2
OH
8
3
7
6
+
N
5
NH2
4
CH2CH3
2-Hydroxyethidium (2-OH-E+)
Menadione-Induced HE Fluorescence in BAEC
A
B
8
**
control
5 M
10 M
Fluorescence intensity
(relative to control)
**p<0.005
**
6
4
**
2
0
20 M
40 M
10 M +
MnTBAP
Menadione-induced HE fluorescence in BAECs. (A) Phase-contrast and fluorescence images of BAECs
treated with different concentrations of menadione for 30 min. After menadione treatment, BAECs were
washed with DPBS and incubated with HE (10 µM) for 20 min. Cells were washed twice with DPBS and
kept in the culture medium. The red fluorescence generated from HE was monitored by using
fluorescence microscopy. BAECs were preincubated with MnTBAP (150 µM) for 2 h. After washing the
cells free of extracellular MnTBAP, they were treated with menadione (10 µM) for 30 min, and HEderived fluorescence was measured. (B) Densitometric analysis of data shown in A.
Zhao et al. PNAS 102:5727-32 (2005)
HPLC identification of 2-OH-E+ from the O2.-/HE reaction in BAECs
E+
Control
2-OH-E+
Menadione (5 M)
HPLC/fluorescence
chromatograms of authentic
2-OH-E+ (labeled 1) and E+
(labeled 2). (Inset) HPLC
peak intensity of 2-OH-E+
and E+ at different
concentrations. BAECs were
treated with various
concentrations of menadione
Menadione (10 M)
Menadione (20 M)
Menadione (40 M)
Menadione (20 M)
+ MnTBAP
25
27
29
Time (min)
31
HPLC Calibration Using
Authentic Compounds
300
y = 14.617x
r = 0.9978
2-OH-Ethidium
250
Ethidium
Peak height (arbitrary unit)
350
200
2-OH-Ethidium (5 M)
150
y = 3.45x
r = 0.996
100
Ethidium (5 M)
50
Concentration (M)
0
0
5
10
15
20
25
25
27
29
Time (min)
31
Intracellular Concentration of 2-OH-E+ and E+
10
4
3
**
**
*
**
8
*
*
2
*p<0.05,
**p<0.005
**
***
6
**
**
**
1
0
4
[ Ethidium ] (M)
[ 2-OH-Ethidium ] (M)
2-OH-Ethidium
Ethidium
2
0
The actual concentrations of 2-OH-E+ and E+ generated under the conditions
described above were calculated by using the calibration data shown
The Effect of SOD on Product Formation
Medium
B
Cells
A
Control
Control
+Menadione
+Menadione
+Menadione
+SOD
25
27
+Menadione
+SOD
29
31
Time (min)
33
35
25
27
29
31
Time (min)
33
35
The effect of SOD on HE/O2.- product formation in BAECs treated with menadione. (A) HPLC
traces of HE-derived products in cell lysates were obtained from control, menadione-treated,
and menadione/SOD-treated cells. BAECs were preincubated with 100 units/ml SOD for 1 h
and then treated with 40 µmol/liter menadione and HE. (B) Experimental conditions were the
same as in A except that HE-derived products were analyzed in the cell culture medium.
Comparison Between HPLC and EPR Analyses
Cell
A
HPLC
B
Menadione
+ HE
Menadione + HE
Menadione
+ HE + BMPO
Menadione
+ HE + BMPO
25
27
Medium
29
31
Time (min)
33
35
25
27
29
31
Time (min)
33
35
H3C
C
EPR
D Menadione + BMPO
Menadione + BMPO
Menadione + BMPO + SOD
10 G
t-BuO2C
Menadione + BMPO + SOD
OH
N
H
O
BMPO-OH
10 G
Comparison between HPLC and EPR spin-trapping detection of O2.- in BAECs treated with menadione. (A Upper) BAECs were
treated with menadione (10 µM) for 1 h, then washed with DPBS and incubated with HE (10 µM) for 20 min. (Lower) BAECs were
preincubated with BMPO (25 mM) and then treated with menadione (10 µM) and HE (10 µM). HE-derived oxidation products were
obtained in cell lysates as described earlier. (B) Experimental conditions were the same as in A except that the HE-derived products
were analyzed in the cell culture medium. (C Upper) BAECs were treated with menadione (10 µM) and BMPO (25 mM) for 1 h. EPR
spectra of cell lysates were recorded. (Lower) Experimental conditions were the same as in Upper except for the presence of SOD
(100 units/ml). (D) Experimental conditions were the same as in C except that EPR spectra of media were analyzed.
HPLC Analysis of Superoxide
Production in Cells
5
[ 2-OH-Ethidium ] (M)
Menadione (5 M)
+ HE (10 M)
Ceramide (20 M)
+ HE (10 M)
Ceramide (20 M)
+ Menadione (5 M)
+ HE (10 M)
25
4
3
2
1
16
2-OH-Ethidium
Ethidium
**p<0.01, *p<0.05 **
*p<0.05,
**p<0.01
**
**
**
**
14
12
10
8
6
4
[ Ethidium ] (M)
HE (10 M)
2
0
30
Time (min)
0
35
HPLC analysis of O2.- production in BAECs treated with menadione and ceramide. (A) HPLC
traces of cell lysates obtained from the control cells and from cells preincubated with
ceramide (20 µM) for 6 h followed by menadione (5 µM) treatment. Cells were washed and
treated with HE (10 µM). (B) The actual concentrations of 2-OH-E+ and E+ peaks calculated
by using the calibration.
Optimal Conditions for Detection by
Fluorescence
Region 3
30
2-OH-Ethidium
Ethidium
80
20
60
40
10
20
0
540
100
560
580
600
620
Wavelength (nm)
640
20
40
10
20
0
560
580
EX 510 nm
2-OH-Ethidium
Ethidium
100
30
80
20
60
40
10
20
0
560
30
60
600
620
Wavelength (nm)
120
0
540
2-OH-Ethidium
Ethidium
80
0
540
0
FI of 2-OH-Ethidium
FI of 2-OH-Ethidium
100
EX 494 nm
Recommended Commonly
used filter
filter
580
600
620
Wavelength (nm)
640
FI of Ethidium
Region 2
FI of 2-OH-Ethidium
Region 1
120
640
FI of Ethidium
EX 490 nm
FI of Ethidium
120
Conclusions
• Because of the overlapping fluorescence spectra
from 2-OH-E+, the “red fluorescence” formed from
HE cannot be used to quantitate intracellular O2∙formation.
• The new HPLC/fluorescence assay using HE as a
probe is more suitable for quantifying intracellular
O2∙-.
• Intracellular formation of 2-OH-E+ is a diagnostic
marker product of O2∙-.
Chemical structures of hydroethidine (HE), Fremy’s salt
(Fs) and nitrosodisulfonate radical dianion (NDS)
Zielonka J, et al. Free Radic Biol Med 39:853-863 (2005)
HPLC Analysis of the HE/Fremy’s Salt Reaction
HPLC analysis of the HE/Fremy’s salt reaction. HE (16 μM) was added to solution containing NDS (32
μM) in phosphate buffer (pH 7.4, 50 mM) and incubated for 60 min. HPLC traces were obtained from
solutions containing E+ (16 μM), HE (16 μM), and 2-OH-ethidium formed from an incubation mixture
containing HE (16 μM), xanthine (1.0 mM), and xanthine oxidase (0.05 U/ml) in phosphate buffer.
Incubation mixtures were analyzed by HPLC with a fluorescence detector using an excitation
wavelength 510 nm and emission wavelength 595 nm. Because of the differences in the fluorescence
intensities of the products analyzed, the scale was set differently for each HPLC trace.
Zielonka J, et al. Free Radic Biol Med 39:853-863 (2005)
Acknowledgements







Hongtao Zhao (MCW)
Henry M. Fales (NIH)
Edward A. Sokoloski (NIH)
Rodney L. Levine (NIH)
Jeannette Vasquez Vivar (MCW)
Joy Joseph (MCW)
Jacek Ziolonka (MCW)