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Hydroxyl radical scavenging ability of bacterioruberin
Pergamon
Radiat. Phys. Chem. Vol. 50, No. 3, pp. 267-269, 1997 © 1997ElsevierScienceLtd. All rights reserved Printed in Great Britain PII: S0969-806X(97)00036-4 0969-806X/97 $17.00+ 0.00
HYDROXYL RADICAL SCAVENGING ABILITY OF BACTERIORUBERIN TAKESHI SAITO, YUKO MIYABE, HIROSHI IDE and OSAMU YAMAMOTO Division of Gene Chemistry, Graduate Department of Gene Science, Faculty of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima 739, Japan (Received 10 November 1996; revised 30 January 1997; accepted 19 February 1997) Abstraet--A red carotenoid pigment, bacterioruberin, was extracted from Rubrobacter radiotolerans. The OH scavenging effect of this pigment was studied using a system of thymine degradation and compared with those of cysteine and I]-carotene. Thymine solution (5 x 10-4 tool/din3 with 0.2% SDS buffered at pH 7.0) was irradiated with 6°Co "t-rays in the presence and absence of the scavengers. We found that the dose reducing factor (5.3) of bacterioruberin is much higher than that (2.5) of cysteine and 13-carotene. © 1997 Elsevier Science Ltd
1. INTRODUCTION
2.2. Preparation of solutions
Rubrobacter radiotolerans is the most radioresistant bacterium (Do = 10 kGy) studied. This bacterium was isolated from muddy water in a radioactive hot spring and originally named Arthrobacter radiotolerans by Yoshinaka et al. (1973) and later reclassified to a new genus as Rubrobacter (red rod) on the basis of its chemical characteristics by Suzuki et al. (1988). This bacterium contains red pigments. The main red pigment has been identified as bacterioruberin which is one of the carotenoids (Saito et al., 1994). The purpose of the present study is to clarify whether or not the existence of this red pigment relates to the radioresistance of R. radiotolerans. It is well known that nucleobases are modified or damaged mainly by OH radicals produced from water irradiated with ionizing radiation and that thymine is the most radiolytically degradable among four nucleobases in DNA (Scholes, 1978; T6oule and Cadet, 1978; von Sonntag, 1987). It is also known that cysteine is a typical OH scavenger (Nakken, 1965) and 13-carotene is an effective 02- quencher (Armstrong and Buchanan, 1978). Using a measuring system of thymine degradation by 6°Co y-irradiation (T6oule and Cadet, 1975; Yamamoto and Fuji, 1985), the protective effect of bacterioruberin was studied and compared with those of cysteine and 13-carotene. 2. MATERIALS AND METHODS
2.1. OH scavengers Bacterioruberin was extracted from R. radiotolerans and purified by the method of Saito et al. (1994). Cysteine and I~-carotene (special grade) were purchased from Wako Chemical Company, Japan.
Thymine aqueous solutions were prepared at a concentration of 5 × 10 -4 mol/dm 3. The solutions also contained 3.7 × 107 Bq/dm 3 [2-~4C]-thymine and 0.2% SDS and were buffered at pH 7.0 with 10-3mol/dm 3 phosphate buffer (in final). The concentration of OH scavengers was 10 mol/dm 3, when added. The solutions were bubbled with N20 for 15 min in order to eliminate hydrated electrons (N20 + e~q --,N_, + OH" + OH - ). -
2.3. Irradiation (TLC)
and
thin-layer
4
chromatograph)'
Irradiation was performed with 6°Co 7-rays at a dose-rate of 20 Gy/min and at 0°C. After irradiation, 50 ttl of the solution was spotted on a TLC plate (Merck silica gel 60 F254, 20 × 20 cm) and developed two-dimensionally with the solvents used by Tgoule and Cadet (1975). The ~4C-radioactivity of each TLC spot was measured with a radiochromatoscanner (Berthod Automatic TLC Analyser LB-2852). 2.4. Measurement of light absorbance In order to determine the decolorization rate of bacterioruberin by irradiation, bacterioruberin solutions (I0- 4 moi/dm 3) containing 0.2% SDS were irradiated with different doses in the presence of thymine (5 × 10-4 mol/dm 3) under N20 atmosphere. Light absorbance was measured at an absorption maximum of 440 nm. 2.5. Calculation of free valency In order to know the free valency of the constituting atoms in a bacterioruberin molecule, ab initio calculation of the frontier electron density
267
Takeshi Saito et al.
268
100
£
50
I
o
10 --
II Fig. 1. Two-dimensional radio chromatogram of thymine (5 x 10 -4 mol/dm 3, comprised of 3.7 x 107 Bq/dm 3 [2-14C]thymine and 0.2% SDS, buffered at pH 7.0) irradiated with 6°Co ),-rays at a dose of 1.5 kGy. Spot 1: thymine, Spot 2: 5-hydroxy-5-methylbarbituric acid, Spot 3: thymine glycol, Spots 4 and 5: unknown dimerized or oxidized products.
[f~ = (CrL~MO)2 + (C, HoMo)2] was p e r f o r m e d with the R H F / S T O - 3 G basis set using the G a u s s i a n 92 p r o g r a m package.
X
50
I
F
I
I
I
2
4
6
8
10
12
Dose (kGy) Fig. 2. Effects of the presence of cysteine, 13-carotene and bacterioruberin (10- 4 mol/dm 3) on thymine degradation by ~Co 7-irradiation. Experiment was repeated three times (SE < 5%). O, Bacterioruberin; [], 13-carotene; A, cysteine; x , control without scavengers.
100 v
E
8O
t,,,,
60
©
40
0
20
0¢,,,
¢,~
O'
t 0
0%
o,. °
2
4
6
\
I
8
10
12
Dose ( k G y ) Fig. 3. Decolorization and decrease in light absorbance (440 nm) of bacterioruberin solution. The 3 4 bacterioruberin solutions (10- 4 mol/dm)containingthymine(10mol/dm3) and 0.2% SDS were irradiated under N20.
269
OH scavenging with bacterioruberin OH
\1/ OH 0.027
0.00137
!
0.081
j
0.13
0.002/5 [ 0.(J28 0 J~;:.11.]87
|
0.13
0(110.[?.14 '
J
I
J
[
1H
OH Fig. 4. Chemical structure of bacterioruberin and free valency of constituting atoms obtained by ab initio calculation of the frontier electron density.
3. RESULTS AND DISCUSSION
Acknowledgements--This work was supported in part by the
Urakami Foundation. Figure 1 shows a radio chromatogram of thymine in aqueous solution irradiated with ~°Co 7-rays at a dose of 1.5 kGy. Under these conditions, 46% of thymine was degraded in the absence of scavengers. Degradation of thymine in the presence of scavengers is shown in Fig. 2. At a concentration of 10-4mol/dm 3 of the scavenger, the radiation dose-reducing factor of bacterioruberin (5.3) was much higher than those of 13-carotene (2.5) and cysteine (2.5). The OH scavenging ability of 13-carotene was the same as that of cysteine. Figure 3 shows the relationship between the radiation dose and decolorization of bacterioruberin without thymine, which was obtained by measuring maximal light absorbance at 440 nm. This result suggests a very high reaction rate of bacterioruberin with OH radicals. Figure 4 shows the chemical structure of bacterioruberin along with the distribution of the free valency of constituting atoms. Double bonds are known to be very reactive with OH radicals (Yamamoto, 1986). The bacterioruberin molecule contains 13 conjugated double bonds, which are much more than the nine conjugated double bonds of the 13-carotene molecule. Besides, the free valency of the double-bonded carbon atoms is higher in the central regions of the molecule than that in the end regions. Therefore, it is concluded that the high OH radical scavenging ability of bacterioruberin may be due to the existence of the highly conjugated double bonds which can efficiently act as an oxidation protector. Thus, bacterioruberin will be able to act as a highly effective OH scavenger in the cell of R. radiotolerans. This may be one of the reasons for the extremely high radiotolerance of R. radiotolerans.
REFERENCES
Armstrong, D. A. and Buchanan, J. D. (1978) Reactions of Of, H20., and other oxidants with sulfhydryl enzymes. Photochem, Photobiol. 28, 743. Nakken, K. F. (1965) Radical scavengers and radioprotection. In Current Topics in Radiation Research (Edited by M. Ebert and A. Howard), Vol. 1, p. 49. North-Holland Publishing Company, Amsterdam. Saito, T., Terato, H. and Yamamoto, O. (1994) Pigments of Rubrobacter radiotolerans. Arch. Microbiol. 162, 414. Scholes, G. (1978) Primary events in the radiolysis of aqueous solutions of nucleic acids and related substances. In Effects of Ionizing Radiation on DNA (Edited by J. H/ittermann, W. K6hnlein, R. T~oule and A. J. Bertinchamps), p. 153. Springer-Verlag, Berlin. Suzuki, K., Collins, M. D., Iijima, E. and Komagata, K. (1988) Chemotaxonomic characterization of a radiotolerant bacterium, ,4rthrobacter radiotolerans: Description of Rubrobacter radiotolerans gen. nov., comb. nov. FEMS Microbiol. Lett. 52, 33. T6oule, R. and Cadet, J. (1975) Comparison of radiolysis products of thymine and thymidine with e.s.r, results. Int. J. Radiat. Biol. 27, 211. T~oule, R. and Cadet, J. (1978) Radiation-induced degradation of the base component in DNA and related substance Final products. In Effects q/ Ionizing Radiation on DNA (Edited by J. Hiittermann, W. K6hnlein, R. T6oule and A. J. Bertinchamps), p. 171. Springer-Verlag, Berlin. von Sonntag, C. (1987) The Chemical Basis oJ Radiation Biology, p. 116. Taylor and Francis, London. Yamamoto, O. (1986) Radiation-induced cross-links between carboxylic acids and compounds of biological interest. Radiat. Res. 108, 158. Yamamoto, O. and Fuji, I. (1985) Degradation of thymine in aqueous solution containing 3HHO. Comparison with 6°Co gamma-radiolysis. J. Radiat. Res. 26, 257. Yoshinaka, T., Yano, K. and Yamaguchi, H. (1973) Isolation of highly radioresistant bacterium. Arthrobacter radiotolerans nov. sp. Agr. Biol. Chem. 37, 2269.
Radiat. Phys. Chem. Vol. 50, No. 3, pp. 267-269, 1997 © 1997ElsevierScienceLtd. All rights reserved Printed in Great Britain PII: S0969-806X(97)00036-4 0969-806X/97 $17.00+ 0.00
HYDROXYL RADICAL SCAVENGING ABILITY OF BACTERIORUBERIN TAKESHI SAITO, YUKO MIYABE, HIROSHI IDE and OSAMU YAMAMOTO Division of Gene Chemistry, Graduate Department of Gene Science, Faculty of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima 739, Japan (Received 10 November 1996; revised 30 January 1997; accepted 19 February 1997) Abstraet--A red carotenoid pigment, bacterioruberin, was extracted from Rubrobacter radiotolerans. The OH scavenging effect of this pigment was studied using a system of thymine degradation and compared with those of cysteine and I]-carotene. Thymine solution (5 x 10-4 tool/din3 with 0.2% SDS buffered at pH 7.0) was irradiated with 6°Co "t-rays in the presence and absence of the scavengers. We found that the dose reducing factor (5.3) of bacterioruberin is much higher than that (2.5) of cysteine and 13-carotene. © 1997 Elsevier Science Ltd
1. INTRODUCTION
2.2. Preparation of solutions
Rubrobacter radiotolerans is the most radioresistant bacterium (Do = 10 kGy) studied. This bacterium was isolated from muddy water in a radioactive hot spring and originally named Arthrobacter radiotolerans by Yoshinaka et al. (1973) and later reclassified to a new genus as Rubrobacter (red rod) on the basis of its chemical characteristics by Suzuki et al. (1988). This bacterium contains red pigments. The main red pigment has been identified as bacterioruberin which is one of the carotenoids (Saito et al., 1994). The purpose of the present study is to clarify whether or not the existence of this red pigment relates to the radioresistance of R. radiotolerans. It is well known that nucleobases are modified or damaged mainly by OH radicals produced from water irradiated with ionizing radiation and that thymine is the most radiolytically degradable among four nucleobases in DNA (Scholes, 1978; T6oule and Cadet, 1978; von Sonntag, 1987). It is also known that cysteine is a typical OH scavenger (Nakken, 1965) and 13-carotene is an effective 02- quencher (Armstrong and Buchanan, 1978). Using a measuring system of thymine degradation by 6°Co y-irradiation (T6oule and Cadet, 1975; Yamamoto and Fuji, 1985), the protective effect of bacterioruberin was studied and compared with those of cysteine and 13-carotene. 2. MATERIALS AND METHODS
2.1. OH scavengers Bacterioruberin was extracted from R. radiotolerans and purified by the method of Saito et al. (1994). Cysteine and I~-carotene (special grade) were purchased from Wako Chemical Company, Japan.
Thymine aqueous solutions were prepared at a concentration of 5 × 10 -4 mol/dm 3. The solutions also contained 3.7 × 107 Bq/dm 3 [2-~4C]-thymine and 0.2% SDS and were buffered at pH 7.0 with 10-3mol/dm 3 phosphate buffer (in final). The concentration of OH scavengers was 10 mol/dm 3, when added. The solutions were bubbled with N20 for 15 min in order to eliminate hydrated electrons (N20 + e~q --,N_, + OH" + OH - ). -
2.3. Irradiation (TLC)
and
thin-layer
4
chromatograph)'
Irradiation was performed with 6°Co 7-rays at a dose-rate of 20 Gy/min and at 0°C. After irradiation, 50 ttl of the solution was spotted on a TLC plate (Merck silica gel 60 F254, 20 × 20 cm) and developed two-dimensionally with the solvents used by Tgoule and Cadet (1975). The ~4C-radioactivity of each TLC spot was measured with a radiochromatoscanner (Berthod Automatic TLC Analyser LB-2852). 2.4. Measurement of light absorbance In order to determine the decolorization rate of bacterioruberin by irradiation, bacterioruberin solutions (I0- 4 moi/dm 3) containing 0.2% SDS were irradiated with different doses in the presence of thymine (5 × 10-4 mol/dm 3) under N20 atmosphere. Light absorbance was measured at an absorption maximum of 440 nm. 2.5. Calculation of free valency In order to know the free valency of the constituting atoms in a bacterioruberin molecule, ab initio calculation of the frontier electron density
267
Takeshi Saito et al.
268
100
£
50
I
o
10 --
II Fig. 1. Two-dimensional radio chromatogram of thymine (5 x 10 -4 mol/dm 3, comprised of 3.7 x 107 Bq/dm 3 [2-14C]thymine and 0.2% SDS, buffered at pH 7.0) irradiated with 6°Co ),-rays at a dose of 1.5 kGy. Spot 1: thymine, Spot 2: 5-hydroxy-5-methylbarbituric acid, Spot 3: thymine glycol, Spots 4 and 5: unknown dimerized or oxidized products.
[f~ = (CrL~MO)2 + (C, HoMo)2] was p e r f o r m e d with the R H F / S T O - 3 G basis set using the G a u s s i a n 92 p r o g r a m package.
X
50
I
F
I
I
I
2
4
6
8
10
12
Dose (kGy) Fig. 2. Effects of the presence of cysteine, 13-carotene and bacterioruberin (10- 4 mol/dm 3) on thymine degradation by ~Co 7-irradiation. Experiment was repeated three times (SE < 5%). O, Bacterioruberin; [], 13-carotene; A, cysteine; x , control without scavengers.
100 v
E
8O
t,,,,
60
©
40
0
20
0¢,,,
¢,~
O'
t 0
0%
o,. °
2
4
6
\
I
8
10
12
Dose ( k G y ) Fig. 3. Decolorization and decrease in light absorbance (440 nm) of bacterioruberin solution. The 3 4 bacterioruberin solutions (10- 4 mol/dm)containingthymine(10mol/dm3) and 0.2% SDS were irradiated under N20.
269
OH scavenging with bacterioruberin OH
\1/ OH 0.027
0.00137
!
0.081
j
0.13
0.002/5 [ 0.(J28 0 J~;:.11.]87
|
0.13
0(110.[?.14 '
J
I
J
[
1H
OH Fig. 4. Chemical structure of bacterioruberin and free valency of constituting atoms obtained by ab initio calculation of the frontier electron density.
3. RESULTS AND DISCUSSION
Acknowledgements--This work was supported in part by the
Urakami Foundation. Figure 1 shows a radio chromatogram of thymine in aqueous solution irradiated with ~°Co 7-rays at a dose of 1.5 kGy. Under these conditions, 46% of thymine was degraded in the absence of scavengers. Degradation of thymine in the presence of scavengers is shown in Fig. 2. At a concentration of 10-4mol/dm 3 of the scavenger, the radiation dose-reducing factor of bacterioruberin (5.3) was much higher than those of 13-carotene (2.5) and cysteine (2.5). The OH scavenging ability of 13-carotene was the same as that of cysteine. Figure 3 shows the relationship between the radiation dose and decolorization of bacterioruberin without thymine, which was obtained by measuring maximal light absorbance at 440 nm. This result suggests a very high reaction rate of bacterioruberin with OH radicals. Figure 4 shows the chemical structure of bacterioruberin along with the distribution of the free valency of constituting atoms. Double bonds are known to be very reactive with OH radicals (Yamamoto, 1986). The bacterioruberin molecule contains 13 conjugated double bonds, which are much more than the nine conjugated double bonds of the 13-carotene molecule. Besides, the free valency of the double-bonded carbon atoms is higher in the central regions of the molecule than that in the end regions. Therefore, it is concluded that the high OH radical scavenging ability of bacterioruberin may be due to the existence of the highly conjugated double bonds which can efficiently act as an oxidation protector. Thus, bacterioruberin will be able to act as a highly effective OH scavenger in the cell of R. radiotolerans. This may be one of the reasons for the extremely high radiotolerance of R. radiotolerans.
REFERENCES
Armstrong, D. A. and Buchanan, J. D. (1978) Reactions of Of, H20., and other oxidants with sulfhydryl enzymes. Photochem, Photobiol. 28, 743. Nakken, K. F. (1965) Radical scavengers and radioprotection. In Current Topics in Radiation Research (Edited by M. Ebert and A. Howard), Vol. 1, p. 49. North-Holland Publishing Company, Amsterdam. Saito, T., Terato, H. and Yamamoto, O. (1994) Pigments of Rubrobacter radiotolerans. Arch. Microbiol. 162, 414. Scholes, G. (1978) Primary events in the radiolysis of aqueous solutions of nucleic acids and related substances. In Effects of Ionizing Radiation on DNA (Edited by J. H/ittermann, W. K6hnlein, R. T~oule and A. J. Bertinchamps), p. 153. Springer-Verlag, Berlin. Suzuki, K., Collins, M. D., Iijima, E. and Komagata, K. (1988) Chemotaxonomic characterization of a radiotolerant bacterium, ,4rthrobacter radiotolerans: Description of Rubrobacter radiotolerans gen. nov., comb. nov. FEMS Microbiol. Lett. 52, 33. T6oule, R. and Cadet, J. (1975) Comparison of radiolysis products of thymine and thymidine with e.s.r, results. Int. J. Radiat. Biol. 27, 211. T~oule, R. and Cadet, J. (1978) Radiation-induced degradation of the base component in DNA and related substance Final products. In Effects q/ Ionizing Radiation on DNA (Edited by J. Hiittermann, W. K6hnlein, R. T6oule and A. J. Bertinchamps), p. 171. Springer-Verlag, Berlin. von Sonntag, C. (1987) The Chemical Basis oJ Radiation Biology, p. 116. Taylor and Francis, London. Yamamoto, O. (1986) Radiation-induced cross-links between carboxylic acids and compounds of biological interest. Radiat. Res. 108, 158. Yamamoto, O. and Fuji, I. (1985) Degradation of thymine in aqueous solution containing 3HHO. Comparison with 6°Co gamma-radiolysis. J. Radiat. Res. 26, 257. Yoshinaka, T., Yano, K. and Yamaguchi, H. (1973) Isolation of highly radioresistant bacterium. Arthrobacter radiotolerans nov. sp. Agr. Biol. Chem. 37, 2269.