Unsere Resultate lassen sich durch folgende H ypo­
these** beschreiben: Durch Absorption von Strah­
lung werden in trockener Ribonuclease statistisch
über das Molekül verteilt Wasserstoffatome (oder
Elektronen)
ausgelöst. Die freigesetzten H-Atome
reagieren vorzugsweise mit 6 bestimmten Am ino­
säuren. Durch Veränderung einer dieser Aminosäu­
ren wird ein RNase-Molekül mit einer Wahrschein­
lichkeit von 0,45 inaktiviert, während in 55% aller
Fälle die enzymatische Aktivität erhalten bleibt.
Dieser hier vorgeschlagene Inaktivierungsmechanismus wird durch den kürzlich erhaltenen Befund ge­
Durch Bestrahlung bei 77 ° K werden fünfmal weni­
ger Aminosäuren pro Dosiseinheit verändert als bei
Zimmertemperatur und im gleichen Verhältnis ver­
ringert sich auch die Inaktivierungsrate. Ob diese
Abnahme auf einer bei tiefen Temperaturen verrin­
gerten Reaktivität der H-Atome beruht oder ob pri­
mär weniger Wasserstoff freigesetzt wird, läßt sich
zum gegenwärtigen Zeitpunkt noch nicht entscheiden.
Ein Experiment zur direkten Bestimmung von Emis­
sions- und Reaktionsgeschwindigkeit von H-Atomen
bei tiefen Temperaturen wird vorbereitet38.
H e r r n P r o f. D r . K . G . Z i m m e r d a n k e n w ir f ü r sein
stützt, wonach atomarer Wasserstoff im Trockenen
fö rd e rn d e s
die enzymatische Aktivität von Ribonuclease sowie
und
die biologische Funktionsfähigkeit von DNS und
Bakteriophagen mit großer Wirksamkeit zerstört37.
* * Hypothese ist hier in der von N . W . T im o fe e ff-R e s s o v s k y
(1966) formulierten Bedeutung gebraucht; Ü bersetzung
bei K. G. Zimmer, Strahlentherapie 134, 161 [1 9 6 7 ].
F rau
In teresse,
U.
B äuerle
H errn
fü r
P ro f.
D r.
D r.
die A u s f ü h r u n g
U.
der
H
agen
M o l.-
G e w .-B e s tim m u n g e n an d e r U lt r a z e n t r ifu g e , d en A s s i ­
stentinnen K . B r a u e r , R . M o d e s t u n d E . K
u s s in g
fü r
s o r g fä lt ig e M ita rb e it.
37 H. Jung u. K . K ü rz in g e r, Radiat. Res., im Druck.
38 K . K ü rz in g e r u. H. Jung, in Vorbereitung.
Light Scattering and Viscosimetric Studies on DNA after the Photoreaction
with Some Furocoumarins
F.
D
a l l
’A
c q u a
,
M.
T
e r b o j e v ic h
,
and F.
B
e n v e n u t o
Istituti di Chimica farmaceutica e di Chim ica organica dell’Universita di Padova —
Centro Nazionale di chimica del Farm aco e dei Prodotti biologicamente attivi
e Centro Nazionale delle macromolecole, Sezioni di Padova, del Consiglio Nazionale delle R icerdie
(Z. Naturforschg. 23 b, 943— 945 [1968] ; eingegangen am 8 . Januar 1968)
The light scattering measurements and the viscosimetric “ i-assay” perform ed on native D N A
solutions before and after irradiation at 3655 A in the presence of furocoumarins show that the
C4-cycloadditions of furocoumarins to the 5,6-double bond of the pyrimidine bases of D N A , which
take place in the photoreactions, produce neither breakages of the polynucleotide chains nor relevant
modifications of the macromolecule conformation.
It is well known that some furocoumarins by ir­
radiation at 3655 Ä are able to produce photo­
sensitizing effects on various biological systems 1.
The more active substances are psoralen and several
its methyl-derivatives2, xanthotoxin, or 8-methoxypsoralen, and bergapten, or 5-methoxy-psoralen.
These substances form complexes with D N A when
added to a solution of the macromolecule3-5 and
by irradiation at 3655 Ä they covalently bind to
the pyrimidine bases of D N A through a photo-C4cyclo-addition reaction in which the 5,6-double bond
of pyrimidines and the double bonds 4,,5/ or 3,4 of
furocoumarins are involved 6.
B e l l i n et al. 7’ 8 have found that other substances,
such as methylene blue and rose bengal, which are
able to photooxidate in a particular manner the
guanine moieties of DNA, produce some breakages
of the polynucleotide chains of the macromolecule.
1 L. M u s a jo and G. R o d ig h ie ro , Experientia [B a se l] 18, 1 5 3
5 F. D a l l ’A c q u a and G . R o d ig h ie ro , Atti Accad. naz. Lincei
40,41 1 [196 6],
6 L . M u s a jo , F. B o rd in , G . C a p o r a le , S. M a r c ia n i and G .
R ig a t t i, Photochem. Photobiol. 6, 711 [196 7].
7 J. S. B e l l i n u. L. I. Grossm an, Photochem. and Photobiol.
4, 45 [196 5],
8 J. S. B e ll i n and G . A. Y an ku s, Biochim. biophysica Acta
[A m sterdam ] 112,363 [196 6].
[1 9 6 2 ],
2 G. C a p o r a le , L . M u s a jo , G. R o d ig h ie ro and F. B a c c ic h e tti,
Experientia [B a s e l], in press.
3 G. R o d ig h ie ro , G. C a p o r a le e T . D o lc h e r , Atti Accad. naz.
Lincei 30, 8 3 [ 1 9 6 1 ] .
4 L. M u s a j o , G. R o d i g h i e r o , G. C o lo m b o , F. T o r l o n e and F.
D a l l ’ A c q a , Experientia [B a se l] 21, 2 2 [ 1 9 6 5 ] .
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The present study was performed with the aim
to ascertain if breakages of the polynucleotide chains
take place as a consequence of the C4-cyelo-addition
of furocoumarins to the pyrimidine bases. W e have
found in light scattering measurements and in viscosimetric experiments valuable arguments showing
that in the photoreaction between furocoumarins
and D N A neither breakages of the polynucleotide
chains nor conformational modifications of the ma­
cromolecule occur.
M aterial and Methods
D N A : The following two samples of DNA were
used:
I: Calf thymus DNA, highly polimerized (Mann
Research Laboratories, New York), T m * = 87°, hypochromicity higher than 37 per cent.
II: Salmon sperm DNA, highly polimerized (Calbiochem, Los Angeles —California), T m * = 86°, hypochromicity higher than 38» per cent.
Furocoumarins. Psoralen and bergapten (5-methoxy-psoralen) were prepared by extraction respecti­
vely from the leaves of Ficus carica 10 and from ber­
gamot o iln . Xanthotoxin (8-methoxy-psoralen) was a
commercial sample purifid by sublimation and by cry­
stallization from ethyl alcohol, mp. 148°.
Irradiation of the solutions. The solutions were
introduced into glass cylindrical containers, 9 cm in
diameter, and irradiated in a thermostatically control­
led room ( + 1 °C ) with a Philips HPW 125 lamp,
which emits almost exclusively at 3655 Ä, at a distance
of 16 cm (0.98 mV/cm2). During the irradiation the
temperature of the solutions did not exceed + 5 °C.
Light scattering measurements. To a DNA (I or II)
0.2% aqueous solution (30 ml) containing NaCl
2-10_ 3 M,was added a methanolic solution of psoralen
(9 mg/ml) to a final concentration of 60 //g/ml of
psoralen (the final concentration of methyl alcohol
was 0.66%).
After having been shaken for 10 minutes, the solu­
tion was irradiated for 90 minutes and then solid NaCl
was added to a 1 M final concentration. From this
solution DNA was precipitated by adding slowly two
volumes of anhydrous ethyl alcohol, washed with 70%
ethyl alcohol and dissolved again into 150 ml of a
0.2 M NaCl aqueous solution.
As a check these operations were performed also on
the same DNA solutions with the addition of psoralen
but not irradiated or with the addition of methyl alco­
hol only (0.66%), without psoralen, and then irradiated.
Other check was made dissolving the samples of DNA
* Determined as M a rm u r and D o ty 9.
9 J. M a r m u r and P. D o ty , J. molecular Biol. 6, 109 [1962].
10 E. S päth and R . H i l l e l , Ber. dtsch. chem. Ges. 72, 1577
[1 9 3 9 ].
11 G. R o d ig h ie ro and G. C a p o r a le , Atti Ist. Veneto Sei. Lettere Arti, Cl. Sei. mat. natur. 112, 97 [1954].
(I and II) directly into 150 ml of aqueous 0.2 M NaCl
solution. A ll these operations were performed in a
thermostatically controlled cold room ( + 1 °C ).
The light scattering determinations, were performed
using a Photometer Sofica Model 42.000 with cylindri­
cal cells immersed in highly purified toluene. The in­
strument was standardized with benzene (clarified with
accuracy) using R90 (5460 Ä) = 16.3 x 10~8 12. The
measurements were made on 0.2 M NaCl aqueous solu­
tions at A = 5460Ä, using the value dn/dc = 0.17 13.
DNA concentrations were determined by spectro­
photometric measurement of the O.D. at 2600 A. The
range of DNA concentrations used was between 10~5
and 10’ 10~5g/ml. All the solutions used were clari­
fied either by filtration through a Millipore SM 5 /u
filter or by prolonged shaking of the solutions with
chloroform-isoamil alcohol mixture 14, followed by cen­
trifugation at 25,000 g for 2 hours.
Viscosimetric measurements (i — assay15). For these
experiments DNA-I 0.05% aqueous solutions con­
taining 10“ 2 M NaCl were used. After filtration
through Millipore SM 5 /u filter, to these were added
concentrated methanolic solutions of furocoumarins
(psoralen, xanthotoxin and bergapten). The final con­
centrations are reported in Table II. As a check, to the
same solution of DNA was added an equal amount of
methyl alcohol, without furocoumarins.
Viscosimetric measurements before and after ir­
radiation were made using an U b b e l h o d e viscosimeter at 25° ±0,05°.
The samples to examine were heated for 15 minutes
at a specified temperature, quenched in ice, reequili­
brated at 25° and the viscosity determined at 25°. This
operation was then repeated at various increasing tem­
peratures of heating (from 25° to 100°). The T’v*,i14
value was calculated for each solution according to
H a m a g u c h i and G e i d u s c h e k 15.
R e s u lts and D isc u ssio n
The results obtained by light scattering measure­
ments m ade on D N A - I not irradiated, or irradiated
alone or in the presence of psoralen are reported in
T able I. V e ry sim ilar results w ere obtained also with
D N A -I I . In F ig. 1 is reported a Zim m
— plot o b ­
tained in one o f these experiments.
The data obtained show that after irradiation in
the presence o f psoralen m olecular weight of D N A
remains unchanged and m oreover that the radius of
gyration rem ains also practically unchanged. These
facts
indicate
that
in the
macromolecule
neither
double chain scissions nor conform ational changes
12 C . I. C a r r e B . H . Zimm, J. chem. Physics 18, 1616 [1 9 5 0 ],
27, 739 [1 9 5 7 ] .
14 G . B e r n a r d i, M akrom olekulare Chem. 72, 205 [1 9 6 4 ].
15 K. H am aguchi and F. P. G eidusch ek, J. Am er. chem. Soc.
84, 1329 [1 9 6 2 ].
13 G . W e i l , J. Polym er Sei.
Therefore if the C4-cycloaddition of furocouma­
rin to the 5,6 double-bond o f a pyrimidine base
F ig. 1. Zimm — plot of D N A irradiated alone
and
irradiated in the presence of psoralen - 0 - 0 —, both precipi­
tated with ethyl alcohol and redissolved in 0.2 M N a C l acqueous solution. Th e experimental conditions are described in
the text.
Sam p le
DNA I
D N A I irrad ia te d in the
absence o f psoralen
D N A I irrad iated in the
presence o f psoralen
M w ■ 10-6
1'R % in Ä
4.55
2440
4.55
4.55
2400
2400
T a b le I. Results of the light-scattering measurements.
occur as a consequence of the photoreaction with
furocoumarins.
However, one can observe that if single chain
scissions occur in only a small number of sites, the
macromolecule can retain its molecular weight and
its conformation.
This fact was observed by B e l l i n and Y a n k u s 8
studying the photooxidative effects of some dyes on
D N A . In the initial periods of irradiation molecular
weight of D N A and viscosity of the solutions re­
mained almost unchanged. However modifications
o f the macromolecule due to infrequent scissions of
a single chain could be detected by using the
“ i-assay” 8’ 15.
In this connection, we point out that M u s a j o et
a l.16 studying the photoreaction between labelled
bergapten and D N A , found that after 2 hours of ir­
radiation, in experimental conditions very similar
to those used in the present experiments 1 molecule
of this furocoumarin was linked with every 154
nucleotides.
Using uniformly tritiated psoralen *, we have
now found that after irradiation in the conditions
indicated above, the ratio between psoralen linked
and nucleotides is 1 :110.
* T h e experimental procedure used for this determination
was similar to that preciously described using — 0 14C H 3
bergapten 1#.
produced a breakage o f the polynucleotide chain,
this breakage would occur with the same frequency
as the C4-cycloaddition, that is 1 in every 110
nucleotides, in the case o f psoralen.
For this reason, besides the light-scattering
measurements, we have also performed the viscosimetric “ i-assay” 15 on D N A after irradiation in the
presence of psoralen, bergapten (o r 5-methoxypsoralen) and xanthotoxin (o r 8-methoxy-psoralen).
The results are reported in Table II. They show
F u ro ­
coum arin
ad d e d
F u ro ­
coum arin
con­
centration
I r r a d ia ­
tion
tim e
O g / m l]
[m in u te s]
__
__
__
—
—
60
psoralen
psoralen
x a n th otox in
x a n th otox in
x an th otox in
b ergap ten
bergap ten
25
25
25
25
25
10
10
—
60
—
60
120
—
60
Ti/2,i
82.5°
82.5°
84.4°
88.9°
84°
88.5°
90°
84.5°
87.5°
A T\/2,\ *
__
+
+
+
+
+
+
+
0
2°
6.4°
1.5°
6°
7.5°
2°
5°
T a b le II. T v*,i values of the D N A solutions with furocoum a­
rins added and irradiated. * In respect of the D N A solution
without furocoumarins and not irradiated.
that the Ti/,,; value o f D N A not only is not decreased
after the irradiation in the presence of furocouma­
rins, as would have happened if breakages of poly­
nucleotide chains had taken place, but on the con­
trary it is slightly increased by adding the furo­
coumarins and even much more so after the ir­
radiation.
These increases of the 7\/t,j value are similar
to those of the Tm value of D N A after irradiation
in the presence of furocoumarins previously ob­
served 17 and indicate a stabilization of the secon­
dary structure of D NA.
After these results we may conclude that the C4cycloadditions of furocoumarins to the 5,6 double
bonds of the pyrimidine bases of D N A , which take
place through the irradiation at 3655 Ä , produce
neither breakages of the polynucleotide chains nor
relevant modifications of the macromolecule con­
formation.
16 L . M u s a j o , G. R o d i g h i e r o , A . B r e c c i a , F. D a l l ’A c q u a and
G. M a l e s a n i , Photodiem. Photobiol. 5 , 739 [1 9 6 6 ].
17 F. D a l l ’ A c q u a and G. R o d i g h i e r o , Rend. A t t i Accad. naz.
Lincei 40, 595 [196 6].