Transcript Document

Biophysical methods analysis on chromatin
Fast neutrons protection of chromatin by Cs+ and Al3+ ions,
analyzed by static spectrofluorimetry and time resolved spectroscopy
Radulescu I, Preoteasa V, Radu L, Serbanescu A, Constantinescu
National Institute of Pathology “Victor Babes”, Bucharest, ROMANIA
1.5
I ( a. u. )
1
2
3
1.2
0.9
0
20
40
60
80
100
Dose ( Gy )
Figure 1. The effect of fast
neutrons irradiation on
the relative fluorescence
intensities of
chromatin - proflavine complexes
(1-without metallic ions, 2-with
Cs+, 3-with Al3+).
I (a.u.)
1.1
The most important effects of the ionizing radiations are the single and double strand breaks (SSB and DSB), modifications
of the DNA bases and deoxyriboses,
well asofthe
occurrence of al
Figure 2. Theaseffect
fast
We taken into account the protective effect offered both by histones, which behave as "scavenger molecules" for OH·_ radicals and by1the high compactness
of
DNA
chromatin;
similar
protec
neutrons
irradiation
on
the
intrinsic
2
In this paper we present a study of the complexes of fast neutrons irradiated chromatin with proflavine.
fluorescence intensities of chromatin
3
Fluorimetric and time resolved spectroscopic determinations (single photon counting method) of chromatin - Pr
0.9complexes were realized.Informations regarding the chromatin proteins damag
tryptophan
(1-without
metallic
ions,
Static fluorescence
2-with Cs+,
Proflavine has different fluorescence characteristics according to the state (bound or unbound to chromatin DNA). The quantum yield of Pr decreases
when 3-with
bindingAP+)
to DNA, due to a charge
Figure 1 presents the relative fluorescence intensities of the complexes of proflavine with irradiated chromatin
0.7 with and without the ions Cs+ and Al3+. An increase in the fluorescence inten
The variation of Pr fluorescence intensities for the same irradiation dose shows different values according to the0 charge
of the60added
20 40
80 ions
100 ( it decreases for Cs+ and becomes almost zero for
Dose (Gy)
Figure 3. Fluorescence decay curve of proflavine free (a) and bound to
The chromatin Trp intrinsic fluorescence intensities decrease with the fast neutrons irradiation dose
(see
fig.
2),
which
suggests
the
protein
damage
within
the
Trp
proximity
chromatin DNA (b). The solid curve shows the computer - calculated
The emission peak shifts toward lower wavelength when the metallic ions are added, suggesting
more obtained
compactfrom
structure
of the chromatin.
This structure
of the chromati
decaya curve
deconvolution
of the experimental
decay with
a double - exponential least - squares fit. The standard errors of the fit for
the lifetime values were presented bellow the decay curves.
Time resolved spectroscopy
The lifetimes of the excited states of Pr ( DNA bound Pr and unbound Pr ) have been determined. For free Pr, a 4.93 ns lifetime was obtained, with a standard
deviation of 0.011. The fluorescence decay profile of Pr, recorded on a time-correlated single photon counting spectrometer, is bi-exponential in the presence of
chromatin (Fig. 3). The fluorescence decays were fitted to single or double exponential functions by the method of iterative convolution. The quality of the fit was
judged by the reduced 2 value (< 1.2), by visual inspection for standard deviations in the weighted residuals and by the Durbin-Watson (DW) parameter.
In the presence of the chromatin, two excited state lifetimes (bound = 1.180.1 ns and unbound = 5.420.05 ns ) are obtained, proving the existence of two
distinct emitting species (Fig. 4). The values obtained for Pr complexed with fast neutrons irradiated chromatin confirm the previous observation that real damage
appears only for high doses (over 60 Gy). One can notice a relevant decrease of the Pr bound state percentage (from 19.5% to 4.9%) and an increase of the Pr
unbound state percentage when the irradiation doses varies from 60 Gy to 100 Gy (Fig. 5). The lifetimes of the two excited states don’t present important changes
for low irradiation doses (0 - 60 Gy). The bound state lifetime decreases for high doses, which indicates a conformational transition of the damaged DNA.
When metallic ions are present, a lower decrease of the Pr bound state percentage with the irradiation dose was observed (Table 2). This is the result of the ion`s
protection, which consists in DNA condensation. It is already concluded that the DNA condensation is due to the influence of the positively charged ions on the
structure and stability of the negatively charged DNA strands.
A greater protective effect for Al3+ compared to the one obtained for Cs+ ions was obtained (Table 1). These results are consistent with the observation that the
radioprotection efficiency increases with the ion`s valence.
By fluorescence determinations, changes of the Pr intercalation parameters in fast neutrons irradiated chromatin DNA have been observed. Fluorescence
techniques provide valuable information on the binding equilibrium, by considering the radiative deexcitation of the complex. The time resolved
spectroscopy measurements have clearly stated a variation of the chromatin - Pr bound percentage and conformational changes in fluorescent marker
proximity, when the chromatin is irradiated with fast neutrons. This indicates chromatin DNA damages produced by irradiation. The damage have been
reduced by the presence of protective factors: metallic ions (Cs+ and Al3+ ).
The single photon counting method proved to be more sensitive than the other ones in assessing chromatin DNA damage. This approach also clarifies
the chromatin - Pr interaction mechanism.
100
t1
4
t2
2
0
0
20
40
60
Dose (Gy)
80
100
Figure 4. The excited state
lifetimes of chromatin proflavine complexes
(t 1-unbound, t 2- bound)
versus fast neutrons dose
Figure 5. The percentage contribution to
the fluorescence of chromatin proflavine complexes (f 1-unbound, f 2bound ) versus fast neutrons dose
80
f%
Lifetime (ns)
6
f1
f2
60
40
20
0
0
20
40
60
Dose (Gy)
80
100