workshop on structure and function of biomolecules, may 13-15, 2004 będelwo, poland
Post on 02-Jan-2016
16 Views
Preview:
DESCRIPTION
TRANSCRIPT
1Institute of Physics of Complex Matter, Ecole Polytechnique Fédérale, CH-1015 Lausanne, Switzerland2The H. Niewodniczański Institute of Nuclear Physics, Polish Academy of Sciences ul. Radzikowskiego 152, 31–342 Kraków, Poland
3Institute of Physics, Polish Academy of Sciences, Al. Lotników 32/46, 02-668 Warsaw, Poland4Laboratoire de Neurobiologie Cellulaire, Ecole Polytechnique Fédérale, CH-1015 Lausanne, Switzerland5Institute of Optoelectronics, Military University of Technology, Kaliskiego Str. 2, 00-908 Warsaw, Poland
Workshop on Structure Workshop on Structure and Function of Biomolecules,and Function of Biomolecules,
May 13-15, 2004May 13-15, 2004Będelwo, PolandBędelwo, Poland
Singlet Oxygen (1g)-mediated Oxidation of Cellular and Subcellular Components: ESR and AFM Assays
Bertrand VilenoBertrand Vileno11,, MaMałłgorzata Lekka gorzata Lekka 11,,22,, Andrzej Sienkiewicz Andrzej Sienkiewicz 1,31,3, Pierre Marcoux, Pierre Marcoux11,, Andrzej J. KulikAndrzej J. Kulik11, Sandor Kasas, Sandor Kasas44, Stefan Catsicas, Stefan Catsicas44, Alfreda Graczyk, Alfreda Graczyk55, , and László Forró and László Forró 11
AcknowledgmentsAcknowledgments
ConclusionsConclusions
ReferencesReferences
2 m
AFM tipindentation range ~ 2 m
cell
AFM tip
light guide PBS buffer containing 1g sensitizer
Petri dishsubstrate
CC6060 : an efficient : an efficient 11gg sensitizersensitizer
High yield of the light-induced population of triplet state (T ~ 1) high yield of 1g generation in organic solvents...
However, C60 is not water-soluble …
Covalent multi-functionalization
C60[>N -CH2CH2(OCH2CH2)2 OCH3]6
C60(OH)nwith: n = 20-28 “C60
6arms”
Alfa Aesar Johnson Mattey GmbH, Germany
Delpeux et al., Eur. Polym. J. V34, N7, p.905, 1998
Covalent multi-functionalization
C60[>N -CH2CH2(OCH2CH2)2 OCH3]6
C60(OH)nwith: n = 20-28 “C60
6arms”
Alfa Aesar Johnson Mattey GmbH, Germany
Delpeux et al., Eur. Polym. J. V34, N7, p.905, 1998
Singlet oxygen (1g), the lowest-energy electronically excited state of molecular oxygen, is implicated in aging,
inflammation and disease processes as part of intracellular milieu of reactive oxygen species (ROS). Recently, 1g-mediated photosensitization reactions have found applications in selective eradication of cancer tissues in
photodynamic therapy (PDT) [1, 2]. Here, we report on Electron Spin Resonance (ESR) and Atomic Force Microscopy (AFM) studies of singlet oxygen (1g)-mediated oxidation of various targets, including proteins and cells.
To generate 1g by a photodynamic process in vitro, we used a novel commercially available photosensitizer,
the water–soluble fullerol C60(OH)n, where n ~ 20-28 (Alfa Aesar, Germany), which was activated by
illumination with white light [3].
IntroductionIntroduction
Integrated ESR signal
280' of illumination + 48 hrs in the dark
Before illumination
Before illumination normalizedfor the "final" number ob spins(folded T4L 131/151 SL)
After 280' of illumination + 48 hrs in the dark
Before illumination (folded T4L 131/151 SL)
FOLDED
DENATURED
DENATURED(before normalization)
DENATURED
FOLDED
ESR signal
Den
atu
rati
on b
y G
dnH
Cl
Den
atu
rati
on b
y S
ingl
et O
xyge
n
Integrated ESR signal
280' of illumination + 48 hrs in the dark
Before illumination
Before illumination normalizedfor the "final" number ob spins(folded T4L 131/151 SL)
After 280' of illumination + 48 hrs in the dark
Before illumination (folded T4L 131/151 SL)
FOLDED
DENATURED
DENATURED(before normalization)
DENATURED
FOLDED
ESR signal
Den
atu
rati
on b
y G
dnH
Cl
Den
atu
rati
on b
y S
ingl
et O
xyge
n
valine 131 cysteine + SL
threonine 151 cysteine + SL
ESR: monitoring of the structural damage to the spin-labeled ESR: monitoring of the structural damage to the spin-labeled proteinsproteins
AFMAFM
0
1
2
0 10
E [
kPa]
PBS PBS + C60
(OH)n
20
Illumination Time [min]
0
1
2
0 4010
E [
kP
a]
PBSPBS + C
60(OH)
n
20
Illumination Time [min]
AFM can yield topography and local elastic properties of cells by measuring the Young’s modulus values with nanometer-scale resolution [5]. AFM technique was used to study the photosensitization-induced oxidative stress on living and glutheraldehyde-fixed cells. Topography and local elastic properties were measured for neurons, keratocytes and two different bladder cells lines exposed to the toxic action of 1g. Singlet oxygen was generated in situ directly under the AFM tip. The
investigated cells were immersed in PBS buffer containing 1.3 mM concentration of C60(OH)n [3].
Exemplary AFM image of the gluteraldehyde–fixed rat primary hippocampal neurons under PBS containing C60(OH)n @ 1.3 mM [3].
AFM topography acquired before illumination (left) andafter 40 minutes of illumination with white light (right).
The dependence of the Young modulus values (E) as a function of the exposure time to the toxic action of singlet oxygen for the glutaraldehyde–fixed (left) and living neurons (right).
Experimental details: Model M5 PSI AFM system, unsharpened Si3N4 gold-coated tip (radius ~50 nm), spring constant 0.1 N/m, contact mode, white light from a halogen source. Acquired: topography and force-distance curves.
Fullerol CFullerol C6060(OH)(OH)nn, where n~20-, where n~20-28, 28, is a potent photo-generator of is a potent photo-generator of 11gg in aqueous solutions…in aqueous solutions…
1S0 + h 1S* 3S* 3S* + 3O2
1S0 + 1g
1
g + TMP-OH TEMPOL
1g +
TMP-OH TEMPOL
(EPR silent) (EPR active)
ISC
OH OH
CH3
O CH3 CH3
CH3 N
H
CH3
CH3 CH3
CH3 N •
Fig.2
0 50 100 150 200 250 300
2.0x107
4.0x107
6.0x107
8.0x107
1.0x108
1.2x108
Partial recovery of the ESR signal intensity(after 48h in the dark)
Fit: Second Order Exponential Decay
Num
ber
of S
pins
~ D
oubl
e In
tegr
ated
ESR
Sig
nal [
A.U
.]
Time of Illumination [min]
The doubly spin-labeled protein, T4 lysozyme with MTSSL spin labels attached at V131C/T151C [6], was used as a target for the toxic action of 1g. C60(OH)n @ 0.5 mM was
used as 1g photosensitizer.
Aqueous solutions containing C60(OH)n and T4 lysozyme (@0.5 mM) were illuminated with visible light. ESR spectra were acquired immediately after each dose of illumination.
During photo-generation of singlet oxygen the MTSSL spin labels are also partially destroyed by free radical reactions. Thus, this approach can also be used to probe the free radical processes accompanying singlet oxygen/ROS generation in bio-oxidative studies [7].
0 50 100 150 200 250 300 350 400 450
3280 3300 3320 3340 3360 3380 3400-0.003
-0.002
-0.001
0.000
0.001
0.002
0.003
TEMPOL ESR signal evolution as a function of illumination time
Oxygen-saturated D2O
+ TMP-OH @ 2.5 * 2.5 10-2
M
+ C60
(OH)n (n = 20-28) @ 5 * 10
-4M
ma=1.48G / cf=3345G[100]gain=1E4 / Q=3000Power=2mW / 10scans
EP
R S
ign
al
[A.U
.]
Magnetic Field [G]
Generation of 1g in oxygenated D
2O
containing: TMP-OH(@ 2.5*10-2M) + C60
(OH)n(@ 5*10-4M)
TE
MP
OL
ES
R S
ign
al In
ten
sity
[A
.U.]
Illumination Time [min]
Actin filaments after 6 min of illumination with white light.
Actin content: 0.1 mg/ml in PBS, in the presence of
C60(OH)n @ 2.5 mM.
Actin filaments before illumination with white light.
Actin content: 0.1 mg/ml in PBS, in the presence of C60(OH)n @ 2.5 mM.
Transmission Electron Microscopy (TEM): evidence of Transmission Electron Microscopy (TEM): evidence of 11gg--mediated oxidative stress on actin fibers in the presence mediated oxidative stress on actin fibers in the presence of Cof C6060(OH)(OH)nn
IR and UV-VIS IR and UV-VIS characterization of characterization of the the water-soluble water-soluble fullerol Cfullerol C6060(OH)(OH)nn
ESR evidences ESR evidences photo-photo-generation of generation of 11gg in the in the presence of presence of CC6060(OH)(OH)nn
200 400 600 800 1000 1200 14000
2
C60
(OH)n @ 0.1 mM in H
2O
Ab
sorb
ance
[A
.U.]
Wavelength [nm]
4500 4000 3500 3000 2500 2000 1500 1000 50065
70
75
80
85
90
95
100
105
C60
(OH)n, n=20-28
Alfa Aesar GmBH, Germany
Attached OH groups
3046 cm-1
3455 cm-1
Tra
nsm
itta
nce
[%
]
Wavenumber [cm-1]
A modified technique combining sample illumination with visible light and cw ESR (introduced first by Y. Lion et al. [4]) was employed for detection of 1g-generation in aqueous solutions of C60(OH)n [3].
1. DeRosa MC, Crutchley RJ. Photosensitized singlet oxygen and its applications. Coord Chem Rev 2002;233-234:351-71. 2. Konan YN, Gurny R, Allemann E. State-of-the-art in the delivery of photosensitzer for photodynamic therapy. J Photochem Photobiol B 2002;66:89:106. 3. Vileno B, Sienkiewicz A, Lekka M, Kulik AJ, Forró L. In vitro assay of singlet oxygen generation in the presence of water-soluble derivatives of C60. Carbon 2004;42:1195-1198. 4. Lion Y, Delmelle M, Van de Vorst A. A new method of detecting singlet oxygen production. Nature 1976;263:442-3. 5. Radmacher M. Measuring the elastic properties of biologicla samples with the AFM. IEEE Eng Me Biol Mag 1997;16(2):47-57. 6. Mchaourab HS, Lietzow MA, Hideg K. Hubbell WL. Biochemistry 1996;35:7692-7704. 7. Singh RJ, Mchaourab HS, Feix JB, Hogg N, Kalyanaraman B. Spin-labeling study of the oxidative damage to low-density lipoprotein. Arch Biochem Biophys 1985;236: 238-251.
This work was partly supported by grants: No. 2–P03B–090–19 (A.S.) of the Polish State Committee for Scientific Research (KBN). The Swiss National Science Foundation is acknowledged for supporting in part this study (B.V.). Dr. H. S. Mchaourab (Vanderbilt University, Nashville, USA) is acknowledged for providing us with spin-labeled T4 lysozyme. The authors acknowledge Dr. A. Verkhovsky (EPFL, Lausanne, Switzerland) for providing keratocytes for this study.
1. This study brings the evidence that the water-soluble fullerol C60(OH)n, with n = 20-28, is an efficient 1g-generator in aqueous media and might be implemented
as oxidizing agent in biological systems. 2. We also show that ESR in combination with site-selective spin labeling might be employed for more detailed study of oxidative stress on biomolecular targets, including proteins.3. We emphasize that AFM can be used as a sensitive tool for detecting early changes occurring to sub-cellular structures of cells that are exposed to the oxidatative stress. Our AFM observations of the changes of the local elastic propertiesof cells point to the actin-rich cortex and focal points as primary targets to the toxic action of 1g.
Control experiment: ESR traces of the intact spin-labeled T4 lysozyme (top), after prolonged illumination in the absence of C60(OH)n (center), and in the presence of C60(OH)n prior to illumination (bottom).
3280 3300 3320 3340 3360 3380 3400
280' + 48 h in the dark
280'180'
60'
100'
40'20'
10'
2'
5'
tlight
= 0'
ES
R S
ign
al [
A.U
.]
Magnetic Field [G]
Chemical denaturation of T4 lysozyme (GdnHCl @ 4 M) (top panels) and light-induced changes to T4 lysozyme in the presence of C60(OH)n @ 0.5 mM (bottom panels).
top related