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Biochemical studies on the induction of chloroplast development in Euglena gracilis
285
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 95653
T H E CHEMICAL FORM OF a2p-COMPOUNDS A F T E R N U C L E A R R E C O I L REACTIONS IN N E U T R O N - I R R A D I A T E D N U C L E O T I D E S AND DNA
M I T S U H I K O A K A B O S H I , T O S H I O M A E D A AND A S A K O W A K I
Research Reactor Institute Kyoto University, Kumatori-eho, Sennan-gun, Osaka (Japan) ( R e c e i v e d D e c e m b e r 2oth, 1966)
SUMMARY
I. Monodeoxynucleotides, deoxynucleotide triphosphates and DNA were irradiated in a pneumatic tube of the K y o t o University reactor and a study was made of ~2p derived from the nuclear reaction 31P(n, 7)3'P. 2. When mononucleotides were irradiated, the largest fraction of the ~ P appeared in phosphorous acid, and the next largest in phosphoric acid; but when nucleotide triphosphates and DNA were irradiated, most of the 32p appearing in inorganic fractions was found in hypophosphorous acid. 3- When DNA was irradiated, organic asP-compounds comprised more than 50 % of the radioactive material; in the case of mononucleotides, the figure was about 20 %, and in the case of nucleotide triphosphates, the organic fraction was small. 4- In irradiated DNA, 32p was released b y phosphatase. This suggests that, after the recoil reaction, 32p recombined with the exposed end of the chain.
INTRODUCTION
Although m a n y studies have been made of the physicochemical effects of the action of ionizing radiations on DNA and related substances 1-3, the effects of irradiating these P-containing substances with neutrons, and especially with thermal neutrons, remain obscure. Recently, MAUTNER and co-workers 4,5 reported that, in neutron-irradiated AMP, ADP, ATP, etc., 32p did not remain in the initial organic compounds. Also, HALMANN AND MILLERe observed changes in viscosity and sedimentation of reactor-irradiated DNA; furthermore, they found that a high proportion of a2p in the DNA molecules could not be released b y treatment with alkaline phosphatase. In spite of these studies, however, most details of the changes caused b y neutron-irradiation of DNA and related substances are still unknown. This paper deals with the results of our attempts to clarify the behavior of 32p in this situation by means of paper chromatography and autoradiography. Biochim. Biophys. Acta, i42 (I967) 2 8 5 - 2 9 I
286
M. AKABOSHI, T. MAEDA, A. WAKI
MATERIALS AND METHODS
Salmon-sperm DNA (supplied b y California Biochemical Co.) and dATP, dCTP, dAMP, dGMP, dCMP and dTMP (supplied by Sigma Co.) were irradiated. Bovine pancreatic deoxyribonuclease I, snake venom phosphodiesterase, wheat germ acid phosphatase and calf mucosa alkaline phosphatase (supplied by Sigma Co.) were used in analysing the irradiation products. Samples (i mg) were sealed into polyethylene tubes. These were all put into the same container and were irradiated for 3o min, unless otherwise stated. Irradiation was carried out in a pneumatic tube of the Kyoto University reactor with a neutron flux of approx. 4 " lO12 n • cm 2. sec-1. Activated samples were allowed to stand for 7 days or more to eliminate disturbing radioactivity due to 24Na (ref. 7). After this, the samples were dissolved in o.5-I.O ml of distilled water, and about IOO pg of each sample was spotted on Toyo Roshi No. 5I-A chromatographic paper (2 × 3o cm). Ascending chromatography was carried out with the isobutyric acid: o.5 M NH4OH (lO:6 v/v) system 8. The activity of spots was determined with a paper-chromatographic analyzer (Aloka PCS-4) and integration of each peak was carried out by weighing the shadow part of the recorded chart. Autoradiograms were taken on Fuji X-ray film. The time of exposure was 2 4 days. The positions of organic compounds on the chromatogram were determined with an ultraviolet lamp (Toshiba FI-3S), and of inorganic P by reactivation followed by autoradiography. RESULTS
Irradiation in the dry state The autoradiograms of some irradiated and chromatographed nucleotides are shown in Fig. I. Monodeoxynucleotides yielded four main spots, three of which were
. . . .
dCTP
Fig. I. A u t o r a d i o g r a m s of nucleotide p r o d u c t s a f t e r i r r a d i a t i o n in t h e d r y s t a t e . A, (1AMP; A', d A M P a f t e r acid p h o s p h a t a s e t r e a t m e n t ; A", d A M P a f t e r p h o s p h o d i e s t e r a s e t r e a t m e n t ; C, dCMP; C', d C M P a f t e r acid p h o s p h a t a s e t r e a t m e n t ; C", d C M P a f t e r p h o s p h o d i e s t e r a s e t r e a t m e n t ; a, 3'2P-organic c o m p o u n d ; b, p h o s p h o r i c acid (/2F, o. i8); c, p h o s p h o r o u s acid (RF, o.25); d, h y p o p h o s p h o r o u s acid (RE, o.45); X2, u n i d e n t i f i e d (RE, o.57); X a, u n i d e n t i f i e d (RF, o.7I ).
t~iochim, ltiophvs..4c1(*, I42 (1967) 2 8 5 - 2 9 i
NEUTRON IRRADIATION OF NUCLEOTIDES AND
DNA
287
identified as inorganic compounds, viz., phosphoric acid, phosphorous acid and hypophosphorous acid; most of the remaining s~p appeared to be bound in organic compounds, since it was released by treatment with acid phosphatase. However, one component of the irradiated materials (spots in position (a) in Fig. I) was not changed by treatment with snake-venom phosphodiesterase on either the paper chromatograph or the autoradiograph. Since the spot did not move from its initial position it could not have been original unchanged material. Moreover, it is considered that the component would have very high specific activity, for the spot could not be detected under ultraviolet light. A similar result was obtained from the analysis of irradiated DNA, but nucleotide triphosphates yielded other spots which could not be identified. The activities of these spots were determined by use of the chromatograph-analyzer and the fraction of the total activity for each spot is shown as a percentage in Table I. TABLE 32p
I
DISTRIBUTION
REVEALED
IN
BY PAPER
VARIOUS
PRODUCTS
CHROMATOGRAPHY
OF
NEUTRON-IRRADIATED
NUCLEOTIDES
AND
DNA
(O.~) O F T O T A L A C T I V I T Y )
a, b, c a n d d r e p r e s e n t the spots i l l u s t r a t e d in F i g . i. T h e r e m a i n i n g a c t i v i t i e s (X 2 and X3) were c a l c u l a t e d t o g e t h e r as e. All v a l u e s are t h e m e a n s of t w o or three s e p a r a t e l y o b t a i n e d values.
Spot
Deoxynucleotide monophosphate
,% b c d e * DNA
Deoxynucleotide triphosphate
DN.4
A
G
C
T
dA T P
dCTP
(i)
(2) *
2I.(~ 16. 4 26.3 14.9 20.8
41.O 17. 4 28.4 lO.9 2. 3
23. 7 22.8 26-5 13-9 13.1
24. 9 22.9 20.4 15.9 16.o
6.2 12. 7 15.7 53.7 11. 7
6.9 11.8 20.0 4 4 .2 17.1
50.7 3.2 lO. 7 3°.0 5.4
23. 5 13.1 16. 7 37 .1 9.7
after acid p h o s p h a t a s e t r e a t m e n t .
For the irradiated mononucleotides, the activities in organic components of the product represented about 2o % of the total activity and remaining activities were found in inorganic components, viz., phosphorous acid, phosphoric acid and hypophosphorous acid, in this order. For the irradiated nucleotide triphosphates, TABLE a2p
II
DISTRIBUTION
IN
NEUTRON-IRRADIATED
DNA
DETERMINED
BY
CHEMICAL
PRECIPITATION
A b o u t 3oo/~g o f D N A w a s irradiated for lO, 3 o a n d 6 o rain in t h e d r y state, and, after being left for 7 days, m a c r o n l o l e c u l a r D N A w a s p r e c i p i t a t e d b y i o % trichloroacetic acid. The s u p e r n a t a n t was b r o u g h t to a l k a l i n i t y and p h o s p h o r i c acid w a s p r e c i p i t a t e d in the presence o f M g 2~ a n d s o d i u n l p h o s p h a t e as carrier. k
Time o/ irradiation (rain)
Total activity counts per rain
lO 3° 6o
648 237 ° 459 °
DNA
n
Phosphoric acid
Remaining activity
counts
% o~
per min
total
counts per rain
% o/ total
counts per rain
% o/ total
47-7 46.5 45-5
72 313 56I
II.1 I3.2 12.2
267 956 i94o
41.2 4o.3 42.3
3o9 IIOI
2089
Biochim. Biophys. Acta, t42 (1967) 285 291
288
M. AKABOSHI, T. MAEDA, A. WAKI
however, the highest activity was found in hypophosphorous acid, and the next highest in phosphorous acid; organic 3ZP-compounds constituted a smaller fraction than in the case of mononucleotides. Irradiated DNA yielded a pattern similar to that for nucleotide triphosphates with regard to inorganic P, although the asp activity in the organic part was nearly 50 % of the total. This high retention of 3~p in DNA was confirmed b y studying the precipitation of DNA by IO % trichloroacetic acid (Table II). When irradiated DNA was treated with acid phosphatase, the major part of the 32p activity disappeared. This shows that the ~2P-atoms were attached to end groups of the DNA chains.
Irradiation in the aqueous state
For each of DNA, dATP, dAMP and dCMP, i mg was dissolved in o.5 ml of distilled water. The solutions were sealed in polyethylene tubes and irradiated under the conditions described above. The results showed that, in spite of the different forms of the starting materials, the dry materials and the solutions yielded quite similar autoradiograms (Fig. 2).
Fig. 2. A u t o r a d i o g r a m s of p r o d u c t s f r o m nucleotides and D N A after irradiation in a q u e o u s solution. a, b, c, d, X 2 a n d X 3 are the s p o t s illustrated in Fig. I. X1 (RF, 0.47) and X 4 (RF, 0.9o) c o u l d n o t be identified.
The most notable spot coming from the material irridiated in solution was found in the position of hypophosphorous acid; the amount of .~2p ill organic fractions, and in phosphoric acid and phosphorous acid, was small in this case. Some clear spots were detected which could not be identified; two of them appeared in the same positions as spots found after the irradiation of nucleotide triphosphates in the dry state. These results are shown in Table I I I . Biochim. Biophys. Acta, i42 (1967) 285-291
NEUTRON IRRADIATION OF NUCLEOTIDES AND
DNA
289
TABLE III a2p DISTRIBUTION IN VARIOUS FRACTIONS OF NEUTRON-IRRADIATED NUCLEOTIDES AND D N A IN A Q U E O U S S O L U T I O N (% O F T O T A L ACTIVITY) All a b b r e v i a t i o n s are as in Figs. I and 2. The r e m a i n i n g activities (X 1 and X4) were calculated as e.
Spot
DNA
dA T P
dAMP
dCM P
a b c d X1 X= e
11.6 2.8 5 .8 44.9 21.1 9.6 4-3
t.i 1.5 3.7 54.4 21. 5 6.9 lO.9
o.8 1.6 4 .1 52.9 17. 7 7.9 I4.9
1. 4 1.8 6.1 54.5 16.9 9.3 9.9
DISCUSSION
Our results obtained from irradiation of materials in the dry state showed that the fate of a 32p atom derived from the nuclear reaction 31p(n, V)a*P depended to some extent on its original environment. Thus, for irradiated mononucleotides, the highest fraction of the total activity was found in phosphorous acid and the next highest in phosphoric acid. However, for nucleotide triphosphates, the highest fraction of the total activity was found in hypophosphorous acid with the next highest fraction in phosphorous acid, and this was the same for DNA except that the activity in the organic fraction was in this case nearly 50 % of the total. One possible hypothesis to explain these facts is as follows. When 31p atoms capture thermal neutrons, the resulting metastable excited 3~p nucleons emit v-rays in falling to the ground state. The total energy of these v-rays is 7.9 MeV (ref. 9). I t is generally accepted that the recoil energy of a 3~p nucleus due to these v-rays would be sufficient to break the chemical bonds around the a2p atom, and various 32p-compounds could then appear from the eventual recombination1°, n. In the present case, the following active atoms or groups m a y be expected as a result of the nuclear reaction: 31P(n, y)32p, viz., 32p*, off,_~", O* and OH*, and possibly also H *,
'32p~
o.y
[ ~+a=p]
o
HO-P=O
OH~
0
(H*)
I
OH HO-P=O
organic - P
phosphoric acid
I
OH OH
HO - 4 = 0
phosphorous acid
I
H H HO--4=O Scheme I.
I H
~p~hosphorousadd
Biochim. Biophys. Acta, 142 (i967) 285-291
29o
M. AKABOSHI, T. MAEDA, A. WAKI
which may come from fast neutron collisions or from the effect of y-rays (see Scheme I). (The asterisks do not necessarily imply radicals, since ion ion recombination reactions may occur.) Since active P has a great affinity for 0 atoms 12 and supposing that a a2p atom, when released from the target substance by recoil, would recombine through a double bond with one O atom, then the residual valencies will be filled by the competitive reaction of other active atoms or groups, the particular species depending on the form of the phosphorus in the original substances. In mononucleotides, the amount of OH* in the system is readily concluded to be larger than in irradiated DNA because of the diester linkage of P in the DNA molecule. The fact that hypophosphoras acid is dominant in irradiated DNA can be easily explained on the basis of this hypothesis. Recoiled 32p from DNA appears to have a two-fold possibility of forming organic 32P-compounds compared with that from mononucleotides (recoiled a2p from DNA will be able to recombine at either the 3' or the 5' position of DNA). This also explains the high recombination in DNA. In the case of nucleotide triphosphates, the third P atom from the 5' position can be regarded as sinfilar to the P atom in mononucleotides, while the other two are not. Therefore, nucleotide triphosphates appear to have more of a DNA-like character than do mononucleotides; thus, it is reasonable that hypophosphorous acid is dominant in irradiated nucleotide triphosphates. Furthermore, in this case, there were less organic ~2P-compounds than in the other cases; this may have been due to the difficulty of reforming the original energy-rich bonds. If the sub.~tances were irradiated in aqueous solution, resultant materials were quite similar irrespective of different species of solute. This, we suppose, was due to the active atoms or groups originating from target substances being overcome by H* and OH" originating from the medium, in the competition to fiI1 the residual valencies of 32P- O. According to HALMANN AND MILLER 6, asp is coupled to the irradiated DNA polymer chain by diester bonds, and cannot be released by alkaline phosphatase. However, our results demonstrated the release of 32p by treatment with acid phosphatase, and, moreover, additional experiments with alkaline phosphatase treatment gave similar results. Thus, we conclude that more than half the 3~p atoms were attached to the 3' or 5' terminals of irradiated DNA, in which situation the phosphatase could easily attack. ACKNOWLEDGEMENTS
We thank Prof. Y. Klso and his co-workers for their kind advice and discussion, Prof. S. IWATAfor his great interest and support for this work, and Miss Y. TANAKA for technical assistance. REFE RENCES 1 P. ALEXANDER, J. T. LETT, H. ~IOROSON AND I£. A. STACEY, Intern. J. Radiation Biol., Suppl., i (I96O) 47. 2 C. PONNAMPERUMA, av{. M. LEMMON AND M. CALVIN, l~adialion Res., 18 (1963) 54 o. 3 1~- COLLYNS, S. OKADA, G. SCHOLES, J. J. \ ¥ E I S S AND C. M. W H E E L E R , R a d i a t i o n Res., 25 (I965) 526.
Biochim. Biophvs. ,4cta, i42 (1967) 285 29 t
NEUTRON IRRADIATION OF NUCLEOTIDES AND DNA
291
4 H. G. MAUTNER, B. DONNELLY, C. M. LEE AND G. W. LEDDICOTTE, J. Am. Chem. Soc., 84 (1962) 202. 5 H. G. MAUTNER, C. M. LEE AND M. H. KRACKOV, J. Am. Chem. Soc., 85 (1963) 245. 6 M. HALMANN AND 1. R. MILLER, Biochim. Biophys. Acta, 72 (1963) 475. 7 M. AKABOSHI, A. \VAKI AND T. MAEDA, Zoo1. Mag. (Tokyo), 75 (1966) 183. 8 B. ~¥[AGASANIC, E. VISCHER, R. DONIGER, D. ELSON AND E. CHARGAFF, J. Biol. Chem., 186 (195o) 37. 9 T. H. BRAID, Phys. Rev., lO2 (1956) 11o9. io I. G. CAMPBELL, A. POCZYNAJILO AND A. SIUDA, J. Inorg. Nucl. Chem., IO (1959) 225. I I M. SHIMA AND S. UTSUM1, J. Inorg. Nucl. Chem., 20 (1961) 177. I2 M. HALMANN, Chem. Rev., 64 (1964).
Biochim. Biophys. Acta, i42 (I967) 285-291
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 95653
T H E CHEMICAL FORM OF a2p-COMPOUNDS A F T E R N U C L E A R R E C O I L REACTIONS IN N E U T R O N - I R R A D I A T E D N U C L E O T I D E S AND DNA
M I T S U H I K O A K A B O S H I , T O S H I O M A E D A AND A S A K O W A K I
Research Reactor Institute Kyoto University, Kumatori-eho, Sennan-gun, Osaka (Japan) ( R e c e i v e d D e c e m b e r 2oth, 1966)
SUMMARY
I. Monodeoxynucleotides, deoxynucleotide triphosphates and DNA were irradiated in a pneumatic tube of the K y o t o University reactor and a study was made of ~2p derived from the nuclear reaction 31P(n, 7)3'P. 2. When mononucleotides were irradiated, the largest fraction of the ~ P appeared in phosphorous acid, and the next largest in phosphoric acid; but when nucleotide triphosphates and DNA were irradiated, most of the 32p appearing in inorganic fractions was found in hypophosphorous acid. 3- When DNA was irradiated, organic asP-compounds comprised more than 50 % of the radioactive material; in the case of mononucleotides, the figure was about 20 %, and in the case of nucleotide triphosphates, the organic fraction was small. 4- In irradiated DNA, 32p was released b y phosphatase. This suggests that, after the recoil reaction, 32p recombined with the exposed end of the chain.
INTRODUCTION
Although m a n y studies have been made of the physicochemical effects of the action of ionizing radiations on DNA and related substances 1-3, the effects of irradiating these P-containing substances with neutrons, and especially with thermal neutrons, remain obscure. Recently, MAUTNER and co-workers 4,5 reported that, in neutron-irradiated AMP, ADP, ATP, etc., 32p did not remain in the initial organic compounds. Also, HALMANN AND MILLERe observed changes in viscosity and sedimentation of reactor-irradiated DNA; furthermore, they found that a high proportion of a2p in the DNA molecules could not be released b y treatment with alkaline phosphatase. In spite of these studies, however, most details of the changes caused b y neutron-irradiation of DNA and related substances are still unknown. This paper deals with the results of our attempts to clarify the behavior of 32p in this situation by means of paper chromatography and autoradiography. Biochim. Biophys. Acta, i42 (I967) 2 8 5 - 2 9 I
286
M. AKABOSHI, T. MAEDA, A. WAKI
MATERIALS AND METHODS
Salmon-sperm DNA (supplied b y California Biochemical Co.) and dATP, dCTP, dAMP, dGMP, dCMP and dTMP (supplied by Sigma Co.) were irradiated. Bovine pancreatic deoxyribonuclease I, snake venom phosphodiesterase, wheat germ acid phosphatase and calf mucosa alkaline phosphatase (supplied by Sigma Co.) were used in analysing the irradiation products. Samples (i mg) were sealed into polyethylene tubes. These were all put into the same container and were irradiated for 3o min, unless otherwise stated. Irradiation was carried out in a pneumatic tube of the Kyoto University reactor with a neutron flux of approx. 4 " lO12 n • cm 2. sec-1. Activated samples were allowed to stand for 7 days or more to eliminate disturbing radioactivity due to 24Na (ref. 7). After this, the samples were dissolved in o.5-I.O ml of distilled water, and about IOO pg of each sample was spotted on Toyo Roshi No. 5I-A chromatographic paper (2 × 3o cm). Ascending chromatography was carried out with the isobutyric acid: o.5 M NH4OH (lO:6 v/v) system 8. The activity of spots was determined with a paper-chromatographic analyzer (Aloka PCS-4) and integration of each peak was carried out by weighing the shadow part of the recorded chart. Autoradiograms were taken on Fuji X-ray film. The time of exposure was 2 4 days. The positions of organic compounds on the chromatogram were determined with an ultraviolet lamp (Toshiba FI-3S), and of inorganic P by reactivation followed by autoradiography. RESULTS
Irradiation in the dry state The autoradiograms of some irradiated and chromatographed nucleotides are shown in Fig. I. Monodeoxynucleotides yielded four main spots, three of which were
. . . .
dCTP
Fig. I. A u t o r a d i o g r a m s of nucleotide p r o d u c t s a f t e r i r r a d i a t i o n in t h e d r y s t a t e . A, (1AMP; A', d A M P a f t e r acid p h o s p h a t a s e t r e a t m e n t ; A", d A M P a f t e r p h o s p h o d i e s t e r a s e t r e a t m e n t ; C, dCMP; C', d C M P a f t e r acid p h o s p h a t a s e t r e a t m e n t ; C", d C M P a f t e r p h o s p h o d i e s t e r a s e t r e a t m e n t ; a, 3'2P-organic c o m p o u n d ; b, p h o s p h o r i c acid (/2F, o. i8); c, p h o s p h o r o u s acid (RF, o.25); d, h y p o p h o s p h o r o u s acid (RE, o.45); X2, u n i d e n t i f i e d (RE, o.57); X a, u n i d e n t i f i e d (RF, o.7I ).
t~iochim, ltiophvs..4c1(*, I42 (1967) 2 8 5 - 2 9 i
NEUTRON IRRADIATION OF NUCLEOTIDES AND
DNA
287
identified as inorganic compounds, viz., phosphoric acid, phosphorous acid and hypophosphorous acid; most of the remaining s~p appeared to be bound in organic compounds, since it was released by treatment with acid phosphatase. However, one component of the irradiated materials (spots in position (a) in Fig. I) was not changed by treatment with snake-venom phosphodiesterase on either the paper chromatograph or the autoradiograph. Since the spot did not move from its initial position it could not have been original unchanged material. Moreover, it is considered that the component would have very high specific activity, for the spot could not be detected under ultraviolet light. A similar result was obtained from the analysis of irradiated DNA, but nucleotide triphosphates yielded other spots which could not be identified. The activities of these spots were determined by use of the chromatograph-analyzer and the fraction of the total activity for each spot is shown as a percentage in Table I. TABLE 32p
I
DISTRIBUTION
REVEALED
IN
BY PAPER
VARIOUS
PRODUCTS
CHROMATOGRAPHY
OF
NEUTRON-IRRADIATED
NUCLEOTIDES
AND
DNA
(O.~) O F T O T A L A C T I V I T Y )
a, b, c a n d d r e p r e s e n t the spots i l l u s t r a t e d in F i g . i. T h e r e m a i n i n g a c t i v i t i e s (X 2 and X3) were c a l c u l a t e d t o g e t h e r as e. All v a l u e s are t h e m e a n s of t w o or three s e p a r a t e l y o b t a i n e d values.
Spot
Deoxynucleotide monophosphate
,% b c d e * DNA
Deoxynucleotide triphosphate
DN.4
A
G
C
T
dA T P
dCTP
(i)
(2) *
2I.(~ 16. 4 26.3 14.9 20.8
41.O 17. 4 28.4 lO.9 2. 3
23. 7 22.8 26-5 13-9 13.1
24. 9 22.9 20.4 15.9 16.o
6.2 12. 7 15.7 53.7 11. 7
6.9 11.8 20.0 4 4 .2 17.1
50.7 3.2 lO. 7 3°.0 5.4
23. 5 13.1 16. 7 37 .1 9.7
after acid p h o s p h a t a s e t r e a t m e n t .
For the irradiated mononucleotides, the activities in organic components of the product represented about 2o % of the total activity and remaining activities were found in inorganic components, viz., phosphorous acid, phosphoric acid and hypophosphorous acid, in this order. For the irradiated nucleotide triphosphates, TABLE a2p
II
DISTRIBUTION
IN
NEUTRON-IRRADIATED
DNA
DETERMINED
BY
CHEMICAL
PRECIPITATION
A b o u t 3oo/~g o f D N A w a s irradiated for lO, 3 o a n d 6 o rain in t h e d r y state, and, after being left for 7 days, m a c r o n l o l e c u l a r D N A w a s p r e c i p i t a t e d b y i o % trichloroacetic acid. The s u p e r n a t a n t was b r o u g h t to a l k a l i n i t y and p h o s p h o r i c acid w a s p r e c i p i t a t e d in the presence o f M g 2~ a n d s o d i u n l p h o s p h a t e as carrier. k
Time o/ irradiation (rain)
Total activity counts per rain
lO 3° 6o
648 237 ° 459 °
DNA
n
Phosphoric acid
Remaining activity
counts
% o~
per min
total
counts per rain
% o/ total
counts per rain
% o/ total
47-7 46.5 45-5
72 313 56I
II.1 I3.2 12.2
267 956 i94o
41.2 4o.3 42.3
3o9 IIOI
2089
Biochim. Biophys. Acta, t42 (1967) 285 291
288
M. AKABOSHI, T. MAEDA, A. WAKI
however, the highest activity was found in hypophosphorous acid, and the next highest in phosphorous acid; organic 3ZP-compounds constituted a smaller fraction than in the case of mononucleotides. Irradiated DNA yielded a pattern similar to that for nucleotide triphosphates with regard to inorganic P, although the asp activity in the organic part was nearly 50 % of the total. This high retention of 3~p in DNA was confirmed b y studying the precipitation of DNA by IO % trichloroacetic acid (Table II). When irradiated DNA was treated with acid phosphatase, the major part of the 32p activity disappeared. This shows that the ~2P-atoms were attached to end groups of the DNA chains.
Irradiation in the aqueous state
For each of DNA, dATP, dAMP and dCMP, i mg was dissolved in o.5 ml of distilled water. The solutions were sealed in polyethylene tubes and irradiated under the conditions described above. The results showed that, in spite of the different forms of the starting materials, the dry materials and the solutions yielded quite similar autoradiograms (Fig. 2).
Fig. 2. A u t o r a d i o g r a m s of p r o d u c t s f r o m nucleotides and D N A after irradiation in a q u e o u s solution. a, b, c, d, X 2 a n d X 3 are the s p o t s illustrated in Fig. I. X1 (RF, 0.47) and X 4 (RF, 0.9o) c o u l d n o t be identified.
The most notable spot coming from the material irridiated in solution was found in the position of hypophosphorous acid; the amount of .~2p ill organic fractions, and in phosphoric acid and phosphorous acid, was small in this case. Some clear spots were detected which could not be identified; two of them appeared in the same positions as spots found after the irradiation of nucleotide triphosphates in the dry state. These results are shown in Table I I I . Biochim. Biophys. Acta, i42 (1967) 285-291
NEUTRON IRRADIATION OF NUCLEOTIDES AND
DNA
289
TABLE III a2p DISTRIBUTION IN VARIOUS FRACTIONS OF NEUTRON-IRRADIATED NUCLEOTIDES AND D N A IN A Q U E O U S S O L U T I O N (% O F T O T A L ACTIVITY) All a b b r e v i a t i o n s are as in Figs. I and 2. The r e m a i n i n g activities (X 1 and X4) were calculated as e.
Spot
DNA
dA T P
dAMP
dCM P
a b c d X1 X= e
11.6 2.8 5 .8 44.9 21.1 9.6 4-3
t.i 1.5 3.7 54.4 21. 5 6.9 lO.9
o.8 1.6 4 .1 52.9 17. 7 7.9 I4.9
1. 4 1.8 6.1 54.5 16.9 9.3 9.9
DISCUSSION
Our results obtained from irradiation of materials in the dry state showed that the fate of a 32p atom derived from the nuclear reaction 31p(n, V)a*P depended to some extent on its original environment. Thus, for irradiated mononucleotides, the highest fraction of the total activity was found in phosphorous acid and the next highest in phosphoric acid. However, for nucleotide triphosphates, the highest fraction of the total activity was found in hypophosphorous acid with the next highest fraction in phosphorous acid, and this was the same for DNA except that the activity in the organic fraction was in this case nearly 50 % of the total. One possible hypothesis to explain these facts is as follows. When 31p atoms capture thermal neutrons, the resulting metastable excited 3~p nucleons emit v-rays in falling to the ground state. The total energy of these v-rays is 7.9 MeV (ref. 9). I t is generally accepted that the recoil energy of a 3~p nucleus due to these v-rays would be sufficient to break the chemical bonds around the a2p atom, and various 32p-compounds could then appear from the eventual recombination1°, n. In the present case, the following active atoms or groups m a y be expected as a result of the nuclear reaction: 31P(n, y)32p, viz., 32p*, off,_~", O* and OH*, and possibly also H *,
'32p~
o.y
[ ~+a=p]
o
HO-P=O
OH~
0
(H*)
I
OH HO-P=O
organic - P
phosphoric acid
I
OH OH
HO - 4 = 0
phosphorous acid
I
H H HO--4=O Scheme I.
I H
~p~hosphorousadd
Biochim. Biophys. Acta, 142 (i967) 285-291
29o
M. AKABOSHI, T. MAEDA, A. WAKI
which may come from fast neutron collisions or from the effect of y-rays (see Scheme I). (The asterisks do not necessarily imply radicals, since ion ion recombination reactions may occur.) Since active P has a great affinity for 0 atoms 12 and supposing that a a2p atom, when released from the target substance by recoil, would recombine through a double bond with one O atom, then the residual valencies will be filled by the competitive reaction of other active atoms or groups, the particular species depending on the form of the phosphorus in the original substances. In mononucleotides, the amount of OH* in the system is readily concluded to be larger than in irradiated DNA because of the diester linkage of P in the DNA molecule. The fact that hypophosphoras acid is dominant in irradiated DNA can be easily explained on the basis of this hypothesis. Recoiled 32p from DNA appears to have a two-fold possibility of forming organic 32P-compounds compared with that from mononucleotides (recoiled a2p from DNA will be able to recombine at either the 3' or the 5' position of DNA). This also explains the high recombination in DNA. In the case of nucleotide triphosphates, the third P atom from the 5' position can be regarded as sinfilar to the P atom in mononucleotides, while the other two are not. Therefore, nucleotide triphosphates appear to have more of a DNA-like character than do mononucleotides; thus, it is reasonable that hypophosphorous acid is dominant in irradiated nucleotide triphosphates. Furthermore, in this case, there were less organic ~2P-compounds than in the other cases; this may have been due to the difficulty of reforming the original energy-rich bonds. If the sub.~tances were irradiated in aqueous solution, resultant materials were quite similar irrespective of different species of solute. This, we suppose, was due to the active atoms or groups originating from target substances being overcome by H* and OH" originating from the medium, in the competition to fiI1 the residual valencies of 32P- O. According to HALMANN AND MILLER 6, asp is coupled to the irradiated DNA polymer chain by diester bonds, and cannot be released by alkaline phosphatase. However, our results demonstrated the release of 32p by treatment with acid phosphatase, and, moreover, additional experiments with alkaline phosphatase treatment gave similar results. Thus, we conclude that more than half the 3~p atoms were attached to the 3' or 5' terminals of irradiated DNA, in which situation the phosphatase could easily attack. ACKNOWLEDGEMENTS
We thank Prof. Y. Klso and his co-workers for their kind advice and discussion, Prof. S. IWATAfor his great interest and support for this work, and Miss Y. TANAKA for technical assistance. REFE RENCES 1 P. ALEXANDER, J. T. LETT, H. ~IOROSON AND I£. A. STACEY, Intern. J. Radiation Biol., Suppl., i (I96O) 47. 2 C. PONNAMPERUMA, av{. M. LEMMON AND M. CALVIN, l~adialion Res., 18 (1963) 54 o. 3 1~- COLLYNS, S. OKADA, G. SCHOLES, J. J. \ ¥ E I S S AND C. M. W H E E L E R , R a d i a t i o n Res., 25 (I965) 526.
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NEUTRON IRRADIATION OF NUCLEOTIDES AND DNA
291
4 H. G. MAUTNER, B. DONNELLY, C. M. LEE AND G. W. LEDDICOTTE, J. Am. Chem. Soc., 84 (1962) 202. 5 H. G. MAUTNER, C. M. LEE AND M. H. KRACKOV, J. Am. Chem. Soc., 85 (1963) 245. 6 M. HALMANN AND 1. R. MILLER, Biochim. Biophys. Acta, 72 (1963) 475. 7 M. AKABOSHI, A. \VAKI AND T. MAEDA, Zoo1. Mag. (Tokyo), 75 (1966) 183. 8 B. ~¥[AGASANIC, E. VISCHER, R. DONIGER, D. ELSON AND E. CHARGAFF, J. Biol. Chem., 186 (195o) 37. 9 T. H. BRAID, Phys. Rev., lO2 (1956) 11o9. io I. G. CAMPBELL, A. POCZYNAJILO AND A. SIUDA, J. Inorg. Nucl. Chem., IO (1959) 225. I I M. SHIMA AND S. UTSUM1, J. Inorg. Nucl. Chem., 20 (1961) 177. I2 M. HALMANN, Chem. Rev., 64 (1964).
Biochim. Biophys. Acta, i42 (I967) 285-291