National Institute of Pathology

1
Biophysical methods analysis on chromatinFast
neutrons protection of chromatin by Cs and Al3
ions, analyzed by static spectrofluorimetry and
time resolved spectroscopyRadulescu I,
Preoteasa V, Radu L, Serbanescu A, Constantinescu

National Institute of Pathology Victor Babes,
Bucharest, ROMANIA
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, as well as the occurrence
of alkali and heat labile sites (revealed as
strand breaks after alkaline or thermic treatment
of irradiated DNA). The ionizing particles can
have either direct effects on the DNA
constituents or indirect effects, mediated by the
OH_ radicals, appeared as products of the water
radiolysis. The occurrence of SSB and DSB in the
chromatin DNA strands is supposed to hinder the
DNA-dye complex formation. Usually, the dyes
present different fluorescence parameters in the
two possible states, so one can correlate the
lifetime or the quantum yield with the extent of
the damage. We taken into account the protective
effect offered both by histones, which behave as
"scavenger molecules" for OH_ radicals and by
the high compactness of DNA chromatin similar
protective effects might be the result of the
metallic ion addition, which triggers some
conformational transitions of the chromatin DNA
toward a highly compacted structure. In this
paper we present a study of the complexes of fast
neutrons irradiated chromatin with
proflavine. Fluorimetric and time resolved
spectroscopic determinations (single photon
counting method) of chromatin - Pr complexes were
realized.Informations regarding the chromatin
proteins damage were obtained by monitoring the
fluorescence of Trp. Static fluorescence Proflav
ine has different fluorescence characteristics
according to the state (bound or unbound to
chromatin DNA). The quantum yield of Pr decreases
when binding to DNA, due to a charge transfer
from adenine to the dye. The result is a lower
quantum yield of DNA-Pr complex, compared to that
of free Pr. Figure 1 presents the relative
fluorescence intensities of the complexes of
proflavine with irradiated chromatin with and
without the ions Cs and Al3. An increase in the
fluorescence intensity, which reflects a lower
bound Pr ratio consecutive to the chromatin DNA
irradiation is observed. This behaviour is
explained by the less binding of Pr to chromatin
damaged DNA. A negative relationship between
bound Pr ratio and the irradiation dose is
observed. An important decrease of the ratio is
produced only for high irradiation doses, due to
the protective role of the histones and of the
high compactness of the DNA within the
chromatin. The variation of Pr fluorescence
intensities for the same irradiation dose shows
different values according to the charge of the
added ions ( it decreases for Cs and becomes
almost zero for Al3 ) (Fig. 1). The protective
effect of metallic ions is observed.
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).

Figure 2. The effect of fast neutrons irradiation
on the intrinsic fluorescence intensities of
chromatin tryptophan (1-without metallic ions,
2-with Cs, 3-with AP)
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 and also
the appearance of a non radiative deexcitation
process. Also, a redshift (about 9 nm) of the
emission peak occurs one can infer a looser
structure of the protein and perhaps of the whole
chromatin after irradiation. The emission peak
shifts toward lower wavelength when the metallic
ions are added, suggesting a more compact
structure of the chromatin. This structure of the
chromatin induced by the metallic ions denotes
also a less uniform distribution of the DNA
within the chromatin (i.e. high DNA concentration
clusters appears). Little variations of Trp
relative fluorescence intensities of irradiated
chromatin in metallic ions presence are produced
(Fig. 2).
Figure 3. Fluorescence decay curve of proflavine
free (a) and bound to chromatin DNA (b). The
solid curve shows the computer - calculated decay
curve obtained from 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 (lt 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
dont 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 ions 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 ions 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.
Figure 5. The percentage contribution to the
fluorescence of chromatin - proflavine complexes
(f 1-unbound, f 2-bound ) versus fast neutrons
dose
Figure 4. The excited state lifetimes of
chromatin - proflavine complexes (t 1-unbound, t
2- bound) versus fast neutrons dose