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Photosensitized inactivation of deoxyribonucleic acid
154
PRELIMINARY NOTES
arginine of the nuclei is present in Fraction I. If indeed part of the keratinoid has been converted into products found in Fractions II and III, which is not unlikely 6, the total keratinoid would represent an even higher percentage of the mass of the nuclei, accounting for even more of the total arginine. The molecular ratio arginine/DNA-phosphoric acid groups in the nuclei has further proved to be about 0.9, while the molecular ratio total basic amino acids/DNAphosphoric acid groups is only slightly higher, i.e. about I.O. Evidently in bovine spermatozoa the arginine-rich keratinoid fulfils one of the roles (i.e. the binding of DNA-phosphoric acid groups) of the soluble histones in the somatic cells. The DNA will probably be nestled against a network of keratinoid threads. This difference between somatic cells and this representative of mammalian spermatozoa is in line with the view that in the former the histones regulate the function of I)NA in protein synthesis, while in spermatozoa DNA has no such function; they only have to transport it in a well protected state. A detailed report of this investigation will be published. This work has been supported by grants from the Foundation for Chemical Research in the Netherlands (S.O.N.).
Laboratory /or Physiological Chemistry, The State University, Utrecht (The Netherlands) 1 2 3 4 ,5 6
R. L. C. E. S. j. p.
ELISABETH BRIL-I~ETERSEN H . G . t{. WESTENBRINIK
D. DALLAM AND L. E. THOMAS, Bioehim. Biophys. Acta, I I (1953) 79E. THOMAS AND D. T. MAYER, Science, IiO (1949) 393A. ZITTLE AND R. A. O'DELL, J. Biol. Chem., 14o (1941 ) 899. SCHRAMM, S. MOORE AND E. J. BIGWOOD, Biochem. J., 57 (1954) 33. L. BANDEMER AND t{. J. EVANS, J. Chromalog., 3 (I96°) 431. (]. •ENDREW, in H. NEURATH AND K. BAILEY, The Proteins, B, Vol. 2, New York, 1954, 906.
Received August I5th, 1963 Biochim. Biophys. Acla, 76 (1963) I 5 2 - I 5 4
PN 6120
Photosensitized inactivation of deoxyribonucleic ocid Though it is possible to inactivate transforming DNA with visible light in the presence of a dye 1, the mechanism of this phenomenon is still unknown. It was recently observed that visible light causes selective destruction of guanine moieties in DNA if methylene blue and oxygen are present 2. But a complicating factor in most photosensitization experiments is the conversion of the dyes by the light used. For this reason we decided to test lumichrome (I), a compound related to riboflavin and completely stable to visible light. Transforming I)NA, isolated from a Bacillus subtilis wild type strain, was very rapidly inactivated by light of a high-pressure mercury lamp (Philips H P 125) if lumichrome was present. Of the nucleotides occurring_in DNA only deoxyguanylic acid (If) was broken down very quickly under the experimental conditions. The Bioehim. Biophys. Acla, 76 (1963) 154-156
PRELIMINARY NOTES
155
course of the reaction was studied spectrophotometrically. The spectrum of deoxyguanylic acid appeared to change through two isosbestic points at 245 and 290 m/z (Fig. I). No reaction was found in a nitrogen atmosphere so we infer that an oxidation of guanine was involved. a
0.75
1
H3C .3c" v
0.5C
\(-'~"% I
I
E
0 ~3
025 II
.300 Wavelength ( rnF )
H H
4()0
Fig, I. Spectral c h a n g e s of d e o x y g u a n y l i c acid (0. 4 #M) u p o n i r r a d i a t i o n w i t h a P h i l i p s H P 125 light source in t h e p r e s e n c e of l u m i c h r o m e (o.o 3/tM) a t a d i s t a n c e of 2o c m (sum s p e c t r a given). C u r v e a, p r i o r to i r r a d i a t i o n ; C u r v e b, a f t e r IO m i n of irradiation; C u r v e c, a f t e r x2o m i n of irradiation.
The same results were found in experiments with riboguanylic acid, guanosine and guanine. B y using DNA containing [8J4C]guanine and [SJ4C]adenine it could be shown that upon irradiation with visible light in the presence of lumichrome only guanine was destroyed in the nucleic acid. It was found possible to convert at least 80 % of the guanine in this way. The irradiation of deoxyguanylic acid in the presence of lumichrome was also studied in a Warburg apparatus in order to determine the amount of oxygen used in the reaction. A solution containing 3 #M deoxyguanylic acid and 0.07 #M lumichrome in 2 ml water was irradiated with visible light. The ogygen consumption was found to be 1.2 molecule per molecule riucleotide. To our surprise, CO,. was formed during the irradiation (0.8 molecule per molecule nucleotide). Further experiments revealed that the irradiation of DNA containing [8-14C]guanine led to the formation of laCO~. This means that during the irradiation of DNA the C-8 atom was eliminated as CO S. On irradiation of deoxyguanylic acid in the presence of lumichrome a yellow photoproduct was formed, that still contained the deoxyribose phosphate group. Biochim. Biophys. Acta, 76 (1963) I 5 4 - I 5 6
I56
PRELIMINARY NOTES
Irradiation of guanine gave also rise to a yellow product, which showed the same spectrum as that obtained from deoxygnanylic acid. This indicated that in both products the N atom at position 9 was still present. The guanine photoproduct was isolated. After removing lumichrome b y extraction with n-butanol the yellow-brown compound was precipitated b y acidifying the solution. This compound contained more than 30 % nitrogen and we are at the moment engaged in the identification. Irradiation of [2-a~C~guanine resulted mainly in the formation of one photoproduct containing the 14C-2 atom. This product, however, appeared not to be identical with the yellow photoproduct mentioned above, Consequently at least two degradation products of the guanine are formed. Other studies 3-7 of photosensitization in this laboratory have led us to the conclusion that lumichrome is probably able to transfer its excitation energy to a triplet level. To check this for the himichrome-sensitized reaction of deoxyguanylic acid, we studied the effect of paramagnetic and diamagnetic ions on the conversion of deoxyguanylic acid. If we are dealing with a triplet-triplet transfer, paragmagnetic ions will slow down the reaction, for these ions shorten the lifetime of excited molecules in the triplet state. TABLE I Added
ion
% inhibition
Co*+ Mn*+ Cuz+ Ni*+ Cd~+
55 45 9° 55 o
Ca 2+ K+
o o
Z n 2+
o
Though oxygen also shortens the lifetime of triplets we were not able to study this effect in our reaction because it is essential for the oxidation. A mixture of 3-IO-*#M lumichrome, 25.io-*/zM deoxyguanylic acid and 3" lO-8 M of the ion, total volume 5 ml, was irradiated with two Philips H P I25 lamps at 20 cm distance for 5 min. The paramagnetic ions Co 2+, Ni *+, Mn 2+ and Cu *+ did slow down the reaction while no effect was observed on adding diamagnetic ions like Cd 2+, Ca 2+, Zn ~+ or K+. This observation is an argument for energy transfer to a triplet level, A himichrome-sensitized reaction was also found in experiments with xanthine, but not with adenine and hypoxanthine.
Biochemical and Biophysical Laboratory, Technological University, Delft (The Netherlands)
j . s . SUSSENBACH w. BERENDS
1 j . S. BELLIN AND G. OSTER, Biochim. Biophys. Acta, 42 (196o) 533. z M. I. SIMON AND H. VAN VUNAKIS, J. Mol. Biol., 4 (1962) 488. 8 j . POSTHOlVIA AI~D W. BERENDS, J. Phys. Chem., 66 (1962) 2547. 4 B . H E N D R I K S AND VV'. BERENDS, Rec. Tray. Chim., 77 (1958) 1455 E. ZONDAGj J. POSTHUMA AND ~vV. BERENDS, Biochim. Biophys. Acta, 39 (196o) 178. 6 j . P0STHrd~IA, Biochim. Biophys. Actor, 41 (196o) 538. J. POSTrltlMA, Biochim. Biophys. Acta, 51 (1961) 392.
Received June I9th , 1963 Biochim. Biophys. Acta, 76 (1963) 154-156
PRELIMINARY NOTES
arginine of the nuclei is present in Fraction I. If indeed part of the keratinoid has been converted into products found in Fractions II and III, which is not unlikely 6, the total keratinoid would represent an even higher percentage of the mass of the nuclei, accounting for even more of the total arginine. The molecular ratio arginine/DNA-phosphoric acid groups in the nuclei has further proved to be about 0.9, while the molecular ratio total basic amino acids/DNAphosphoric acid groups is only slightly higher, i.e. about I.O. Evidently in bovine spermatozoa the arginine-rich keratinoid fulfils one of the roles (i.e. the binding of DNA-phosphoric acid groups) of the soluble histones in the somatic cells. The DNA will probably be nestled against a network of keratinoid threads. This difference between somatic cells and this representative of mammalian spermatozoa is in line with the view that in the former the histones regulate the function of I)NA in protein synthesis, while in spermatozoa DNA has no such function; they only have to transport it in a well protected state. A detailed report of this investigation will be published. This work has been supported by grants from the Foundation for Chemical Research in the Netherlands (S.O.N.).
Laboratory /or Physiological Chemistry, The State University, Utrecht (The Netherlands) 1 2 3 4 ,5 6
R. L. C. E. S. j. p.
ELISABETH BRIL-I~ETERSEN H . G . t{. WESTENBRINIK
D. DALLAM AND L. E. THOMAS, Bioehim. Biophys. Acta, I I (1953) 79E. THOMAS AND D. T. MAYER, Science, IiO (1949) 393A. ZITTLE AND R. A. O'DELL, J. Biol. Chem., 14o (1941 ) 899. SCHRAMM, S. MOORE AND E. J. BIGWOOD, Biochem. J., 57 (1954) 33. L. BANDEMER AND t{. J. EVANS, J. Chromalog., 3 (I96°) 431. (]. •ENDREW, in H. NEURATH AND K. BAILEY, The Proteins, B, Vol. 2, New York, 1954, 906.
Received August I5th, 1963 Biochim. Biophys. Acla, 76 (1963) I 5 2 - I 5 4
PN 6120
Photosensitized inactivation of deoxyribonucleic ocid Though it is possible to inactivate transforming DNA with visible light in the presence of a dye 1, the mechanism of this phenomenon is still unknown. It was recently observed that visible light causes selective destruction of guanine moieties in DNA if methylene blue and oxygen are present 2. But a complicating factor in most photosensitization experiments is the conversion of the dyes by the light used. For this reason we decided to test lumichrome (I), a compound related to riboflavin and completely stable to visible light. Transforming I)NA, isolated from a Bacillus subtilis wild type strain, was very rapidly inactivated by light of a high-pressure mercury lamp (Philips H P 125) if lumichrome was present. Of the nucleotides occurring_in DNA only deoxyguanylic acid (If) was broken down very quickly under the experimental conditions. The Bioehim. Biophys. Acla, 76 (1963) 154-156
PRELIMINARY NOTES
155
course of the reaction was studied spectrophotometrically. The spectrum of deoxyguanylic acid appeared to change through two isosbestic points at 245 and 290 m/z (Fig. I). No reaction was found in a nitrogen atmosphere so we infer that an oxidation of guanine was involved. a
0.75
1
H3C .3c" v
0.5C
\(-'~"% I
I
E
0 ~3
025 II
.300 Wavelength ( rnF )
H H
4()0
Fig, I. Spectral c h a n g e s of d e o x y g u a n y l i c acid (0. 4 #M) u p o n i r r a d i a t i o n w i t h a P h i l i p s H P 125 light source in t h e p r e s e n c e of l u m i c h r o m e (o.o 3/tM) a t a d i s t a n c e of 2o c m (sum s p e c t r a given). C u r v e a, p r i o r to i r r a d i a t i o n ; C u r v e b, a f t e r IO m i n of irradiation; C u r v e c, a f t e r x2o m i n of irradiation.
The same results were found in experiments with riboguanylic acid, guanosine and guanine. B y using DNA containing [8J4C]guanine and [SJ4C]adenine it could be shown that upon irradiation with visible light in the presence of lumichrome only guanine was destroyed in the nucleic acid. It was found possible to convert at least 80 % of the guanine in this way. The irradiation of deoxyguanylic acid in the presence of lumichrome was also studied in a Warburg apparatus in order to determine the amount of oxygen used in the reaction. A solution containing 3 #M deoxyguanylic acid and 0.07 #M lumichrome in 2 ml water was irradiated with visible light. The ogygen consumption was found to be 1.2 molecule per molecule riucleotide. To our surprise, CO,. was formed during the irradiation (0.8 molecule per molecule nucleotide). Further experiments revealed that the irradiation of DNA containing [8-14C]guanine led to the formation of laCO~. This means that during the irradiation of DNA the C-8 atom was eliminated as CO S. On irradiation of deoxyguanylic acid in the presence of lumichrome a yellow photoproduct was formed, that still contained the deoxyribose phosphate group. Biochim. Biophys. Acta, 76 (1963) I 5 4 - I 5 6
I56
PRELIMINARY NOTES
Irradiation of guanine gave also rise to a yellow product, which showed the same spectrum as that obtained from deoxygnanylic acid. This indicated that in both products the N atom at position 9 was still present. The guanine photoproduct was isolated. After removing lumichrome b y extraction with n-butanol the yellow-brown compound was precipitated b y acidifying the solution. This compound contained more than 30 % nitrogen and we are at the moment engaged in the identification. Irradiation of [2-a~C~guanine resulted mainly in the formation of one photoproduct containing the 14C-2 atom. This product, however, appeared not to be identical with the yellow photoproduct mentioned above, Consequently at least two degradation products of the guanine are formed. Other studies 3-7 of photosensitization in this laboratory have led us to the conclusion that lumichrome is probably able to transfer its excitation energy to a triplet level. To check this for the himichrome-sensitized reaction of deoxyguanylic acid, we studied the effect of paramagnetic and diamagnetic ions on the conversion of deoxyguanylic acid. If we are dealing with a triplet-triplet transfer, paragmagnetic ions will slow down the reaction, for these ions shorten the lifetime of excited molecules in the triplet state. TABLE I Added
ion
% inhibition
Co*+ Mn*+ Cuz+ Ni*+ Cd~+
55 45 9° 55 o
Ca 2+ K+
o o
Z n 2+
o
Though oxygen also shortens the lifetime of triplets we were not able to study this effect in our reaction because it is essential for the oxidation. A mixture of 3-IO-*#M lumichrome, 25.io-*/zM deoxyguanylic acid and 3" lO-8 M of the ion, total volume 5 ml, was irradiated with two Philips H P I25 lamps at 20 cm distance for 5 min. The paramagnetic ions Co 2+, Ni *+, Mn 2+ and Cu *+ did slow down the reaction while no effect was observed on adding diamagnetic ions like Cd 2+, Ca 2+, Zn ~+ or K+. This observation is an argument for energy transfer to a triplet level, A himichrome-sensitized reaction was also found in experiments with xanthine, but not with adenine and hypoxanthine.
Biochemical and Biophysical Laboratory, Technological University, Delft (The Netherlands)
j . s . SUSSENBACH w. BERENDS
1 j . S. BELLIN AND G. OSTER, Biochim. Biophys. Acta, 42 (196o) 533. z M. I. SIMON AND H. VAN VUNAKIS, J. Mol. Biol., 4 (1962) 488. 8 j . POSTHOlVIA AI~D W. BERENDS, J. Phys. Chem., 66 (1962) 2547. 4 B . H E N D R I K S AND VV'. BERENDS, Rec. Tray. Chim., 77 (1958) 1455 E. ZONDAGj J. POSTHUMA AND ~vV. BERENDS, Biochim. Biophys. Acta, 39 (196o) 178. 6 j . P0STHrd~IA, Biochim. Biophys. Actor, 41 (196o) 538. J. POSTrltlMA, Biochim. Biophys. Acta, 51 (1961) 392.
Received June I9th , 1963 Biochim. Biophys. Acta, 76 (1963) 154-156