ARCHIVES
Vol.
OF BIOCHEMISTRY
277, No. 1, February
AND
BIOPHYSICS
15, pp. 74-79,199O
Evidence for Superoxide Dismutase and Catalase in Mollicutes and Release of Reactive Oxygen Species Beate Meier’
and Gerhard
Chemisches Institut
Received
April
21,1989,
G. Habermehl
der Tieriirztlichen
and in revised
Hochschule,
form
September
Bischofsholer
15, D-3000 Hannover
1, Federal
Republic of Germany
25,1989
The presence of superoxide dismutase was demonstrated in 21 strains of mollicutes, including achuloplasmas, mycoplasmas, and ureaplasmas. No superoxide dismutase activities or only traces were detectable in fresh prepared cell lysates, whereas activities were evident after dialysis of the cell lysates. A further increase in superoxide dismutase activities was observed after the cell lysates were heated to 65°C for 30 s. This might be due to the destruction of enzymatic reactions interfering with the activity tests. Additionally, catalase activities were demonstrated in nearly 50% of the cell lysates, whereas no peroxidase activities were detectable. The production of 0; and HzOz with glucose as substrate was demonstrated for 8 of 10 strains tested. No correlation to the pathogenicities of the strains was indicated. Anaerobic mycoplasmas showed the highest amount of radical production, whereas superoxide dismutase and catalase activities were in the range of activities estimated for aerobic mollicutes. 0 1990 Academic Press, Inc.
Susceptibility and tolerance of organisms toward oxygen are determined by the balance between the production of reactive oxygen species and defense mechanisms in the cell. SOD’ (EC 1.15.1.1), CAT (EC 1.11.1.6), and POD (EC 1.11.1.7) are enzymes that protect against oxygen toxicity. Among them SOD is thought to be indispensable for aerobic organisms in reducing oxygen (1). Only three cases of living aerobic bacteria consuming oxygen but totally lacking SOD have been reported: some strains of lactic acid bacteria (2, 3), some strains of Neisseria gonorrhoeae (4,5), and some strains of myi To whom correspondence should be addressed. 2 Abbreviations used: SOD, superoxide dismutase; CAT, POD, peroxidase; PBS, phosphate-buffered saline; DMPO, methylpyrroline-N-oxide; TEMED, N,N,N’,N’-tetramethylethylenediamine. 74
Damm
catalase; 5,5’-di-
coplasmas and ureaplasma urealyticum (6-9). Those SOD-lacking strains of lactic acid bacteria incorporate up to 25 mmol/liter Mn2+ instead of pmol/liter SOD, which catalyzes the dismutation of 0;. The strains of Neisseriagonorrhoeae containing no SOD are exceptionally rich in CAT, which is insensitive to 0, and POD. Moreover, they possessa cell envelope which is impermeable to OZ. On the other hand mycoplasmas often lack CAT, but they are not extremely sensitive toward reactive oxygen species. The case is quite the reverse; the virulence of some mycoplasmas may be due to reactive oxygen species secreted by these organisms (10-20). Mollicutes are the smallest procaryotic organisms capable of in vitro cultivation; the smallest units of these organisms are only 200 to 300 nm in diameter, with a genome size ranging from 0.5 X 1O’to 1.0 X 10’ Da. Unlike bacteria, they lack a cell wall and often parasitize mammalian cells by attacking the plasma membrane. Some of them are the causative agents of infectious diseases like pleuropneumonia, urogenital infections, and arthritis (21). The present paper reports on studies of several strains of mollicutes that were investigated for the presence of SOD, CAT, POD, and the release of reactive oxygen species. MATERIALS
AND
METHODS
All culture media were obtained from Difco Laboratories (Detroit, MI); cytochrome c (grade III), xanthine oxidase (gradeIII), horseradish peroxidase (type III), and Cu-Zn-SOD were from Sigma (Deisenhoven, FRG); scopoletin, nitroblue tetrazolium, o-dianisidine, and riboflavin were from Serva (Heidelberg, FRG); 5,5’-dimethylpyrrolineN-oxide (DMPO) was from Aldrich (Steinheim, FRG); penicillin and phosphate-buffered saline (PBS) were from Boehringer (Mannheim, FRG). All other reagents used were obtained from Merck (Darmstadt, FRG). Acholeplmma equifetule (Institut fur Mikrobiologie und Tierseuchen, Hannover, FRG), A. hip&on (IMT), A. laidhwii PG8 (National Institutes of Health, Bethesda, MD), Mycoplmma agahctiae (International Reference Center for Animal Mycoplasmas Arhus, 0003-9861/90 Copyright All rights
$3.00 0 1990 by Academic Press, of reproduction in any form
Inc. reserved.
SUPEROXIDE
DISMUTASE
Denmark), M. arthritidis ISR 1 (Professor G. Laber, Sandoz Research Institute, Vienna), d4. bouigenitalium PG 11 (NIH), M. bouis Vonetta (IRC), M. canis PG 14 (NIH), M. capricolum California Kid (IRC), M. capricolum (IRC), M. columbinum (IMT), M. equigenitalium (IMT), M. felis CO (IRC), M. hyorhinis (BTS7) (NIH), M. ouipneumoniae (IRC), M. pulmonis (NIH), M. sub&dun (IMT), and‘ureaplasma sp. a and sp. b (IFM) were propagated in modified Friis medium (22). M. testudinis 01008 (MRC) and the anaerobic strains M. sp. a and M. sp. b (IFM) were cultured in the medium of Chanock et al. (23) at 37°C in a l- to 2-liter volume for 2 to 5 days depending on the species. The cells were sedimented by centrifugation at 25,000g for 25 min at 4°C. The mollicute cell pellet was washed twice with PBS and resuspended in a small volume of this buffer on ice. The identity of each mollicute species was confirmed by using the growth inhibition test (24) and by epi-immunofluorescence (25). For determination of SOD, CAT, and POD, cell lysates were prepared by sonication (10 min, 50% duty cycle with 100 W in ice; Branson Sonic Power Co., Danbury). The aerobic bacterium Erysipelothrin rhusiopathiae T28 was used as control organism in the SOD and CAT activity tests. The bacterium was kindly provided by Professor K. Petzold, Tieriirztliche Hochschule, Hannover. Protein was determined by the Lowry method (26) using bovine serum albumin as standard. SOD was assayed and the units were defined by the xanthine oxidase-cytochrome c assay (27). CAT was assayed by following the absorption of 20 mM H202 at 240 nm and by following the O2 evolution of H202 polarographically with a Clark-type oxygen electrode in a l-ml cell (Kipp & Zonen Delft, The Netherlands). POD activity was tested by catalyzing the reduction of o-dianisidine (28) or scopoletin by H,O, (29). Polyacrylamide gel electrophoresis was performed as described (30) on flat gels of 0.2 mm, and the gels were stained for SOD activity with riboflavin-nitroblue tetrazolium (31) with slight modifications: The nitroblue tetrazolium concentration was reduced to lo-” mol/liter, the amount of iV,N,N’,N’-tetramethylethylenediamine (TEMED) was reduced to 20%, and the riboflavin concentration was reduced to 50%. The gels were stained by illumination with a frosted bulb of 40 W at a distance of 30 cm. The 0; and H,O, production of fresh isolated mollicutes was assayed in PBS supplemented with 25 mmol/liter glucose photometrically at 550 nm by the reduction of acetylated cytochrome c (SOD inhibital part) with a Kontron photometer (32) or fluorometrically with scopoletin (29) using a Kontron fluorometer (Hannover, FRG). The excitation wavelength was 381 nm and the emission wavelength 436 nm. Calibrations were done with hydrogen peroxide. Additionally, the time course of the 0; and H,O, formation was followed by the determination of the luminol and lucigenin chemiluminescence with a six-channel Biolumat LB 9505 (Berthold, Wildbad, FRG) (33). The nature of the radical was identified by ESR spin trapping with DMPO. DMPO was purified by filtration through charcoal (34). The experiments were carried out in a flat cell of 200 ~1 at room temperature with an X-band cavity (Bruker-Analytic B-ER 420, Karlsruhe, FRG). The mollicutes were suspended in PBS supplemented with 25 mmol/liter glucose and 50 mmol/liter DMPO and measured under the following conditions: amplitude, 100 kHz; field modulation, 0.5 mT; microwave power, 150 mW; receiver gain, 4 X 106; recording time, 500 s with a response time of 0.2 s; field center, 0.342 T, sweep width, 20 mT. Only the center (10 mT) was recorded. The magnetic field was measured with a nuclear magnetic resonance oscillator.
RESULTS
In fresh prepared, undialyzed cell lysates of the mollicutes no SOD activity or only traces were detectable with the cytochrome c assay (27). SOD activity was observable after the cell homogenate was dialyzed twice for 12 h against 1000 vol PBS at 4°C. These results lead to
IN
MOLLICUTES
75
the conclusion that the inability to detect SOD activity in the cell lysates by the classical cytochrome c test might be due to a disturbance of the test system. When the mollicute lysates were heated to 65°C for 30 s in PBS, all species showed an SOD activity comparable to those of other aerobic microorganisms. SODS are rather stable enzymes in comparison with enzymatic systems, which might interfere with the test system, such as electron transporting particles of the bacterial respiration chain, and which rapidly lose activity on heating or even storage, To confirm this assumption, old mollicute lysates, which were stored frozen over a period of 1 to 3 years at -2O”C, were tested for SOD activity with the cytochrome c assay. In contrast to freshly prepared cell lysates, SOD activity was detectable in some preparations, according to the heterogeneity of the material. After these samples were heated, they exhibited exactly the same SOD activity as freshly prepared mollicute lysates treated in the same way. These results are summarized in Table I. To confirm that the SOD activity was due to a highmolecular-weight protein and not to low-molecularweight transition metal complexes simulating SOD activity, M. arthritidis lysates were separated by filtration through an ultrafiltration membrane (~30 kDa) and both the low-molecular-weight filtrate and the high-molecular-weight residue were tested for activity. SOD activity remained completely in the filtration residue, and no activity was detectable in the filtrate. By precipitation with (NH&SOI two fractions with SOD activity were obtained: a minor fraction between 65 and 80% (NH&SO4 saturation and the main fraction between 85 and 100% (NH&SO4 saturation. An activity staining on acrylamide gels of some mollicutes for SOD is shown in Fig. 1. To determine the kind of metal present in the active center of the SODS, we tested the SOD activity after treatment of the bacterial extracts with CN-, Hz02, or N, (35). A preincubation of the gels before activity staining for 15 min with CN- (1 mmol/liter), CN- (1 mmol/liter), and HzOz (1 mmol/liter), or NY (4 mmol/liter) caused no inhibition of the SOD. Additionally we determined the SOD activities with the cytochrome c assay (27) after incubation of the bacterial extracts with HzOz (1 mmol/ liter) for 10 min and removal of the H,O, with CAT (10 pmol/liter), as well as in the presence of N, (4 mmol/ liter). The SOD activities were not inhibited by the addition of H202 and decreased less than 30% in the presence of N,. Therefore the mollicutes should contain Mn-SOD. CAT activity was detectable only polarographically, not photometrically, possibly due to the low extinction coefficient of Hz02. The distribution of CAT was heterogeneous among the mollicute species (Table I). CAT appeared to be much more dependent on the culture condi-
76
MEIER
AND
HABERMEHL
TABLE
Activity of Superoxide Dismutase
I
and
Catalase
in Several
Mollicutes
SOD”
CAT*
Species
Fresh”
Dialyzedd
agalactiae arthritidis booigenitalium bovis canis capricolum columbinum equigenitalium felis hyorhinis (BTS7) M. hyorhinis M. ovipneumoniae M. pulmonis M. testudinis Anaerobe M. sp.a Anaerobe M. sp.b Anaerobe M. sp.b A. equifetale A. hippikon A. laidlawii U. sp.a U. sp.b Erysipelothrix rhusiopathiae
ntg 0 0 nt nt Traces 0 nt Traces
nt 10.1 i 2.1 8.3 + 1.9 nt nt 15.4 + 2.3 9.7 + 2.1 nt 17.2 t 3.1
0 18.4 11.7 20.8 0 23.4 0 0 35.7
f0 f 1.4 * 1.0 k 2.1 f 0.2 rk 1.9 k 0.2 *o +- 2.4
18.0 26.7 25.8 22.4 17.0 27.0 25.3 33.4 35.1
f + f k f + 3t f +
0.9 1.3 1.3 1.2 0.8 1.4 1.2 1.7 1.7
nt 31.9 _t 9.3 0 kO.2 0 f0.2 nt 0 f0 nt 0 I?0 0 +o
nt nt nt Traces 0 0 0 0 nt nt Traces nt nt
nt nt nt 14.8 f 9.4 + 12.5 ? 10.1 * 9.3 f nt nt 25.4 k nt nt
19.0 15.0 0 13.8 0 27.0 25.9 17.1 42.0 18.6 14.8 14.6 13.0
+- 1.1 * 0.9 k 0.4 i 1.0 fO.l f 1.7 f 1.7 + 1.3 k 2.2 + 1.2 k 0.9 + 1.0 f 0.9
22.0 21.4 13.8 23.1 24.9 28.7 26.2 24.8 40.1 23.6 36.8 14.3 17.2
* f f ? zk + k + + + + + +
1.1 1.1 0.8 1.2 1.3 1.6 1.5 1.4 1.9 1.3 1.8 0.9 1.0
nt nt nt 1.4 i 0.5 nt 0 2k 0.0 11.7 f 2.7 0 k 0.0 14.4 f 1.1 15.2 + 0.7 8.4 I? 1.5 nt nt
19.7 + 1.5
22.9 If- 1.7
M. M. M. M. M. M. M. M. M. M.
6.2 k 1.3
Old
1.9 2.3 1.4 1.7 2.7
2.9
14.6 k 1.9
12.1 f 1.2
Heated’
Dialyzedd
Note. The mean values and standard deviations of six different determinations are presented. a SOD was determined with cytochrome c (units/mg) (27). * CAT was determined polarographically with Hz02 (nkat). ’ Freshly prepared cell lysates were used for the determination. d Freshly prepared cell lysates were dialyzed twice for 12 h against 1000 vol PBS at 4°C. e Old cell lysates which were stored between 1 and 3 years at -20°C were used for the determination. ‘Cell lysates (freshly prepared and old) in PBS were heated to 60°C for 30 s. g Not tested.
tions than SOD. Within two different cultures of the anaerobic mycoplasma strain sp. b, one culture exhibited CAT activity comparable to that of aerobic mycoplasmas, whereas in the other no catalase activity was detectable. Peroxidase activity was not detectable with either odianisidine or scopoletin. The sensitivity of the scopoletin test was about 1 rig/liter horseradish peroxidase (29). Ten mollicute species, M. arthritidis, M. bovigenitalium, M. capricolum, M. columbinum, M. felis, M. pulmonis, M, testudinis, A. laidlawii, and the anaerobic mycoplasmas sp. a and sp. b were tested for 0; production with lucigenin and for HzOz production with luminol (33) in PBS supplemented with 25 mmol/liter glucose at 37°C (Fig. 2). Light emission was prevented by the addition of SOD (1 pmollliter) and CAT (10 pmol/liter). Without glucose as substrate, no production of 0, and HzOz was detectable. The anaerobic mycoplasmas showed a significantly higher production of reactive oxy-
gen species than the aerobic species. While the production of reactive oxygen compounds by the aerobic mollicutes decreased after some minutes, the anaerobic strains liberated continuously reactive oxygen products for up to 1 h. Equivalent results were obtained in a quantitative assay using acetylated cytochrome c for determination of 0; (32) and scopoletin for determination of the HzOz production (29). Both reactions were prevented by the addition of SOD (1 pmol/liter) or CAT (10 pmol/ liter). Additionally, small amounts of 0; and Hz02 production were detected in M. pulmonis. No production of reactive oxygen species by M. capricolum, M. columbinum, and M. felis was observed with glucose as substrate. The primary radical produced was 0, as demonstrated by ESR spin trapping (34) (Fig. 3). The mollicutes showed decreasing amplitudes of the DMPO-OOH adduct from the anaerobic M. sp. a, to M. sp. b, M. testudinis, which is identical to A. laidlawii, M. bovigenetalium, and M. arthritidis, whereas the hyperfine split-
SUPEROXIDE
4
DISMUTASE
5
6
IN
8
7
77
MOLLICUTES
9
lo
II
12
13
-
FIG. 1. SOD activity staining for several mollicutes. Activity staining for SOD was performed on 7.5% a&amide gels with nitroblue tetrazolium-riboflavin (31). Cell lysates (100 ~1) of the following bacteria were separated: 1, Erys$elothrix rhusiopathiue-aerobic control organism (5.76 mg protein/ml); 2, M. equigenitalium (2.5 mg protein/ml); 3, M. hyorhinis (2.65 mg protein/ml); 4, M. pulmonis (8.5 mg protein/ml); 5, M. bouigenitalium (3.8 mg protein/ml); 6, A. laidlawii (4.6 mg protein/ml); 7, M. hyorhinis (1.2 mg protein/ml); 8, M. felis (3.0 mg protein/ml); 9, M. canis (0.8 mg protein/ml); 10, M. bouis (1.8 mg protein/ml); 11, M. anaerobe sp. b (2.1 mg protein/ml); 12, M. anaerobe sp. a (7.2 mg protein/ml); 13, M. arthritidis (2.3 mg protein/ml).
ting of the radical adduct was not altered. The addition of 1 pmol/liter Cu-Zn-SOD prevented the formation of the radical adducts. DISCUSSION
The present investigations indicate that mollicutes, including some strains of mycoplasmas and ureaplas-
mas, contain SODS, probably with Mn in the active center. In nondialyzed cell extracts no SOD activities or only traces were detectable, and activities were detectable only after dialysis of the cell lysates. This is a wellknown phenomenon. Several organisms were first described as lacking SOD, but later SOD was demonstrated in these organ-
I
6
0
10
20
30 time (mini
40
50
60
i
0
b
2
FIG. 2. Determination of the 0; and H202 production by several mollicutes. (a) Time course nescence with lucigenine (33): 1, M. anaerobe sp. a, 2. M. anaerobe sp. b, 3. M. testudinis = A. (b) Time course of the HzOz production determined by chemiluminescence with luminol(33): bouigenitalium, 4, A. laidlawii, 5. M. testudinis, 6, M. arthritidis. Samples were incubated with at 37°C.
4
6 time hid
8
10
12
of the 0, production determined by chemilumiluidlawii, 4, M. bouigenitulium, 5, M. arthritidis. 1, M. anaerobe sp. a, 2, M. anaerobe sp. b, 3, M. the reagents in PBS with 25 mmol/liter glucose
78
MEIER
AND
isms too (36-41). An increase in total SOD activity compared to that of the cell-free extracts after some preliminary purification steps was also observed in some cases (41-46). SOD activity is often not detectable in bacterial cell-free extracts, especially in those of anaerobes (unpublished data of this laboratory) and has been ascribed to a production of 0, by membrane fragments still present in the bacterial lysates, thus artificially minimizing the real SOD activity (46, 47). We assume that analogous enzymatic mechanisms interfering with the test systems caused the failure to detect SOD in the cell lysates of the mollicutes. By dialyzing, the lysates were liberated from low-molecular-weight substrates for O;producing enzymes. In fact 0; production was observed in lysates of M. pneumoniue (6). Both membranes and cytoplasma caused an SOD inhibital reduction of cytochrome c attributed to a NAD(P)H-flavin oxidase. The presence of SOD in mycoplasmas might be heterogeneous, as in lactic acid bacteria and strains of Nelsseria gonorrhoeae. In crude extracts of A. laidlawii only traces of SOD activity were detectable, whereas in dialyzed and heat-treated extracts the activity was comparable to the results described by Lynch and Cole (7). Three of the thirteen strains tested for CAT activities were assayed previously by Lynch and Cole (7), who demonstrated the absence of CAT. We detected low CAT activities in M. arthritidis, M. pulmonis and A. laidZuwii by following the liberation of O2 from Hz02 polarographically. Like this group, we could not detect any CAT activity by determining the change of HzOz absorption at 240 nm. Additionally, we demonstrated CAT activity for three other strains tested, while in seven other strains tested no CAT activities were detectable.
TABLE Determination
II
of 0; and Hz02 Production
Species M. arthritidis M. bovigenitalum M. capricolum M. columbinum M. felis M. pulmonis M. testudinis Anaerobe M. sp.a Anaerobe M. sp.b A. laidluwii
0,” 1,860 3,733 19 3 0 261 5,740 18,110 10,030 5,268
by Mollicutes HzOz *
f f * zk + !I + * + +
15.6 47.3 0.5 0.0 0.0 13.0 43.0 67.0 59.0 23.0
283 f 930 f 0 + 0 f 0 f 39+ 1185 -t 3750 2 2363 t 1317 r
9.0 22.1 0.0 0.0 0.0 1.2 18.3 51.0 69.0 34.0
Note. The mean values and standard deviations of six different determinations are presented. a 0, production was determined photometrically .at 550 nm by the reduction of acetylated cytochrome c (32) over a period of 30 min at 37°C (nmol/30 min x mg protein). *HZ02 production was determined fluorometrically with scopoletin (excitation 381 nm, emission 436 nm) (29) over a period of 30 min at room temperature, 22°C (nmol/30 min X mg protein).
HABERMEHL
t g =2.0047
ImT FIG. 3.
Radical production by M. anaerobe sp. a was determined by ESR-spin trapping. (a) M. anaerobe sp. a, with a protein concentration of 1 mg/ml, was incubated for 15 min with DMPO at room temperature, 22°C (34). Measuring conditions are described under Materials and Methods. (b) M. anaerobe sp. a was incubated in the presence of 1 pmollliter Cu-Zn-SOD as described above.
In a comparison of these results, it should be always kept in mind that the induction of CAT is usually much more dependent on the culture conditions than that of SOD. Therefore, within two different cultures of an anaerobic mycoplasma strain, CAT was lacking in one and present in the other. Reports on the presence of CAT activity in M. pneumoniae, a human pathogenic species, differ, too. While most reports described a lack of catalase in M. pneumoniae (7, 11, 16, 49) in some cases low CAT activities were determined (8,50-52). Although a weak correlation between CAT activity and the production of reactive oxygen compounds was observed, there were exceptions, the anaerobic mycoplasma strains sp. a and sp. b. While the production of 0, and Hz02 was more than twice the amounts liberated by aerobic mollicutes, the strains either lacked CAT activity or the activity was comparable to that of the aerobic mollicutes. SOD activities were also comparable to those in aerobic mollicutes. It might be speculated that “anaerobiosis” is due to an immoderate liberation of reactive oxygen compounds causing a “relative” deficiency of protective enzymes. With regard to the mollicutes as mostly aerobic organisms, the detection of SOD supports the theory that SOD acts as an obligate antioxidative enzyme. A correlation between the amounts of liberated reactive oxygen compounds and pathogenicity was not observed. The two anaerobic mycoplasma strains A. laidlawii and M. testudinis (Tables I and II) are not pathogenic but liberate 0, and H202. However, it is remarkable that those strains which failed to liberate reactive oxygen compounds were apathogenic. H202 might be one of the pathogenic factors, but the conclusion that an HzOz-producing organism should be pathogenic or that pathogenic organisms should liberate H202 is certainly incorrect. ACKNOWLEDGMENTS This work Ha 457/38-l.
was supported by Deutsche Forschungsgemeinschaft AZ. We thank Professor H. Kirchhoff for kindly providing
SUPEROXIDE
DISMUTASE
the mollicutes, Mrs. R. Schmidt for culturing these organisms, Dipl. Biol. M. Runge for culturing M. arthritidis, and Professor K. Petzold for kindly providing Erysipelothrix rhusiopathiae. Moreover, we gratefully appreciate the opportunities to perform chemiluminescence at the laboratories of Professor W. Leibold and ESR spectroscopy at the University of Bremen.
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51,
Zmmun.
631-641. them.
25. Barile, M. F., and DelGuidice, plasmas, a Ciba Foundation sterdam.
33,151-153.
1. Fridovich, I. (1981) in Oxygen and Living Processes D. L., Ed.), pp. 250-272, Springer-Verlag, New York. 2. Archibald, F. S., and Fridovich, I. (1981) J. Bacterial.
6. Kirby,
79
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6055. 28. Guidotti,
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(1979) The New York.
Mycoplasmas,
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L., and Barile,
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Vet. Microbial.
M. F. (1962)
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L., and
Rotilio,
47. Gregory, E. M., andDrapper, C. E. (1983) Arch. Biochem. 220,293-300. 48. Britton, L., Malinowski, D. P., and Fridovich, I. (1978) riol. 134,229-236. 49. Freundt, E. A. (1958) gaard, Copenhagen.
50. Rodwell, 30. 51. Lecce,
92,958-965.
B., Barra, D., Bossa, F., Calabrese, J. Biol. Chem. 257, 13,977-13,980.
The
Mycoplasmataceae,
A. W., and Rodwell, J. G., and Morton,
52. VanDeMark,
P. J. (1967)
E. S. (1954)
H. E. (1954) Ann.
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N. Y. Acad.
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pp. 57-62. Aust.
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67,62-68.
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Vol.
OF BIOCHEMISTRY
277, No. 1, February
AND
BIOPHYSICS
15, pp. 74-79,199O
Evidence for Superoxide Dismutase and Catalase in Mollicutes and Release of Reactive Oxygen Species Beate Meier’
and Gerhard
Chemisches Institut
Received
April
21,1989,
G. Habermehl
der Tieriirztlichen
and in revised
Hochschule,
form
September
Bischofsholer
15, D-3000 Hannover
1, Federal
Republic of Germany
25,1989
The presence of superoxide dismutase was demonstrated in 21 strains of mollicutes, including achuloplasmas, mycoplasmas, and ureaplasmas. No superoxide dismutase activities or only traces were detectable in fresh prepared cell lysates, whereas activities were evident after dialysis of the cell lysates. A further increase in superoxide dismutase activities was observed after the cell lysates were heated to 65°C for 30 s. This might be due to the destruction of enzymatic reactions interfering with the activity tests. Additionally, catalase activities were demonstrated in nearly 50% of the cell lysates, whereas no peroxidase activities were detectable. The production of 0; and HzOz with glucose as substrate was demonstrated for 8 of 10 strains tested. No correlation to the pathogenicities of the strains was indicated. Anaerobic mycoplasmas showed the highest amount of radical production, whereas superoxide dismutase and catalase activities were in the range of activities estimated for aerobic mollicutes. 0 1990 Academic Press, Inc.
Susceptibility and tolerance of organisms toward oxygen are determined by the balance between the production of reactive oxygen species and defense mechanisms in the cell. SOD’ (EC 1.15.1.1), CAT (EC 1.11.1.6), and POD (EC 1.11.1.7) are enzymes that protect against oxygen toxicity. Among them SOD is thought to be indispensable for aerobic organisms in reducing oxygen (1). Only three cases of living aerobic bacteria consuming oxygen but totally lacking SOD have been reported: some strains of lactic acid bacteria (2, 3), some strains of Neisseria gonorrhoeae (4,5), and some strains of myi To whom correspondence should be addressed. 2 Abbreviations used: SOD, superoxide dismutase; CAT, POD, peroxidase; PBS, phosphate-buffered saline; DMPO, methylpyrroline-N-oxide; TEMED, N,N,N’,N’-tetramethylethylenediamine. 74
Damm
catalase; 5,5’-di-
coplasmas and ureaplasma urealyticum (6-9). Those SOD-lacking strains of lactic acid bacteria incorporate up to 25 mmol/liter Mn2+ instead of pmol/liter SOD, which catalyzes the dismutation of 0;. The strains of Neisseriagonorrhoeae containing no SOD are exceptionally rich in CAT, which is insensitive to 0, and POD. Moreover, they possessa cell envelope which is impermeable to OZ. On the other hand mycoplasmas often lack CAT, but they are not extremely sensitive toward reactive oxygen species. The case is quite the reverse; the virulence of some mycoplasmas may be due to reactive oxygen species secreted by these organisms (10-20). Mollicutes are the smallest procaryotic organisms capable of in vitro cultivation; the smallest units of these organisms are only 200 to 300 nm in diameter, with a genome size ranging from 0.5 X 1O’to 1.0 X 10’ Da. Unlike bacteria, they lack a cell wall and often parasitize mammalian cells by attacking the plasma membrane. Some of them are the causative agents of infectious diseases like pleuropneumonia, urogenital infections, and arthritis (21). The present paper reports on studies of several strains of mollicutes that were investigated for the presence of SOD, CAT, POD, and the release of reactive oxygen species. MATERIALS
AND
METHODS
All culture media were obtained from Difco Laboratories (Detroit, MI); cytochrome c (grade III), xanthine oxidase (gradeIII), horseradish peroxidase (type III), and Cu-Zn-SOD were from Sigma (Deisenhoven, FRG); scopoletin, nitroblue tetrazolium, o-dianisidine, and riboflavin were from Serva (Heidelberg, FRG); 5,5’-dimethylpyrrolineN-oxide (DMPO) was from Aldrich (Steinheim, FRG); penicillin and phosphate-buffered saline (PBS) were from Boehringer (Mannheim, FRG). All other reagents used were obtained from Merck (Darmstadt, FRG). Acholeplmma equifetule (Institut fur Mikrobiologie und Tierseuchen, Hannover, FRG), A. hip&on (IMT), A. laidhwii PG8 (National Institutes of Health, Bethesda, MD), Mycoplmma agahctiae (International Reference Center for Animal Mycoplasmas Arhus, 0003-9861/90 Copyright All rights
$3.00 0 1990 by Academic Press, of reproduction in any form
Inc. reserved.
SUPEROXIDE
DISMUTASE
Denmark), M. arthritidis ISR 1 (Professor G. Laber, Sandoz Research Institute, Vienna), d4. bouigenitalium PG 11 (NIH), M. bouis Vonetta (IRC), M. canis PG 14 (NIH), M. capricolum California Kid (IRC), M. capricolum (IRC), M. columbinum (IMT), M. equigenitalium (IMT), M. felis CO (IRC), M. hyorhinis (BTS7) (NIH), M. ouipneumoniae (IRC), M. pulmonis (NIH), M. sub&dun (IMT), and‘ureaplasma sp. a and sp. b (IFM) were propagated in modified Friis medium (22). M. testudinis 01008 (MRC) and the anaerobic strains M. sp. a and M. sp. b (IFM) were cultured in the medium of Chanock et al. (23) at 37°C in a l- to 2-liter volume for 2 to 5 days depending on the species. The cells were sedimented by centrifugation at 25,000g for 25 min at 4°C. The mollicute cell pellet was washed twice with PBS and resuspended in a small volume of this buffer on ice. The identity of each mollicute species was confirmed by using the growth inhibition test (24) and by epi-immunofluorescence (25). For determination of SOD, CAT, and POD, cell lysates were prepared by sonication (10 min, 50% duty cycle with 100 W in ice; Branson Sonic Power Co., Danbury). The aerobic bacterium Erysipelothrin rhusiopathiae T28 was used as control organism in the SOD and CAT activity tests. The bacterium was kindly provided by Professor K. Petzold, Tieriirztliche Hochschule, Hannover. Protein was determined by the Lowry method (26) using bovine serum albumin as standard. SOD was assayed and the units were defined by the xanthine oxidase-cytochrome c assay (27). CAT was assayed by following the absorption of 20 mM H202 at 240 nm and by following the O2 evolution of H202 polarographically with a Clark-type oxygen electrode in a l-ml cell (Kipp & Zonen Delft, The Netherlands). POD activity was tested by catalyzing the reduction of o-dianisidine (28) or scopoletin by H,O, (29). Polyacrylamide gel electrophoresis was performed as described (30) on flat gels of 0.2 mm, and the gels were stained for SOD activity with riboflavin-nitroblue tetrazolium (31) with slight modifications: The nitroblue tetrazolium concentration was reduced to lo-” mol/liter, the amount of iV,N,N’,N’-tetramethylethylenediamine (TEMED) was reduced to 20%, and the riboflavin concentration was reduced to 50%. The gels were stained by illumination with a frosted bulb of 40 W at a distance of 30 cm. The 0; and H,O, production of fresh isolated mollicutes was assayed in PBS supplemented with 25 mmol/liter glucose photometrically at 550 nm by the reduction of acetylated cytochrome c (SOD inhibital part) with a Kontron photometer (32) or fluorometrically with scopoletin (29) using a Kontron fluorometer (Hannover, FRG). The excitation wavelength was 381 nm and the emission wavelength 436 nm. Calibrations were done with hydrogen peroxide. Additionally, the time course of the 0; and H,O, formation was followed by the determination of the luminol and lucigenin chemiluminescence with a six-channel Biolumat LB 9505 (Berthold, Wildbad, FRG) (33). The nature of the radical was identified by ESR spin trapping with DMPO. DMPO was purified by filtration through charcoal (34). The experiments were carried out in a flat cell of 200 ~1 at room temperature with an X-band cavity (Bruker-Analytic B-ER 420, Karlsruhe, FRG). The mollicutes were suspended in PBS supplemented with 25 mmol/liter glucose and 50 mmol/liter DMPO and measured under the following conditions: amplitude, 100 kHz; field modulation, 0.5 mT; microwave power, 150 mW; receiver gain, 4 X 106; recording time, 500 s with a response time of 0.2 s; field center, 0.342 T, sweep width, 20 mT. Only the center (10 mT) was recorded. The magnetic field was measured with a nuclear magnetic resonance oscillator.
RESULTS
In fresh prepared, undialyzed cell lysates of the mollicutes no SOD activity or only traces were detectable with the cytochrome c assay (27). SOD activity was observable after the cell homogenate was dialyzed twice for 12 h against 1000 vol PBS at 4°C. These results lead to
IN
MOLLICUTES
75
the conclusion that the inability to detect SOD activity in the cell lysates by the classical cytochrome c test might be due to a disturbance of the test system. When the mollicute lysates were heated to 65°C for 30 s in PBS, all species showed an SOD activity comparable to those of other aerobic microorganisms. SODS are rather stable enzymes in comparison with enzymatic systems, which might interfere with the test system, such as electron transporting particles of the bacterial respiration chain, and which rapidly lose activity on heating or even storage, To confirm this assumption, old mollicute lysates, which were stored frozen over a period of 1 to 3 years at -2O”C, were tested for SOD activity with the cytochrome c assay. In contrast to freshly prepared cell lysates, SOD activity was detectable in some preparations, according to the heterogeneity of the material. After these samples were heated, they exhibited exactly the same SOD activity as freshly prepared mollicute lysates treated in the same way. These results are summarized in Table I. To confirm that the SOD activity was due to a highmolecular-weight protein and not to low-molecularweight transition metal complexes simulating SOD activity, M. arthritidis lysates were separated by filtration through an ultrafiltration membrane (~30 kDa) and both the low-molecular-weight filtrate and the high-molecular-weight residue were tested for activity. SOD activity remained completely in the filtration residue, and no activity was detectable in the filtrate. By precipitation with (NH&SOI two fractions with SOD activity were obtained: a minor fraction between 65 and 80% (NH&SO4 saturation and the main fraction between 85 and 100% (NH&SO4 saturation. An activity staining on acrylamide gels of some mollicutes for SOD is shown in Fig. 1. To determine the kind of metal present in the active center of the SODS, we tested the SOD activity after treatment of the bacterial extracts with CN-, Hz02, or N, (35). A preincubation of the gels before activity staining for 15 min with CN- (1 mmol/liter), CN- (1 mmol/liter), and HzOz (1 mmol/liter), or NY (4 mmol/liter) caused no inhibition of the SOD. Additionally we determined the SOD activities with the cytochrome c assay (27) after incubation of the bacterial extracts with HzOz (1 mmol/ liter) for 10 min and removal of the H,O, with CAT (10 pmol/liter), as well as in the presence of N, (4 mmol/ liter). The SOD activities were not inhibited by the addition of H202 and decreased less than 30% in the presence of N,. Therefore the mollicutes should contain Mn-SOD. CAT activity was detectable only polarographically, not photometrically, possibly due to the low extinction coefficient of Hz02. The distribution of CAT was heterogeneous among the mollicute species (Table I). CAT appeared to be much more dependent on the culture condi-
76
MEIER
AND
HABERMEHL
TABLE
Activity of Superoxide Dismutase
I
and
Catalase
in Several
Mollicutes
SOD”
CAT*
Species
Fresh”
Dialyzedd
agalactiae arthritidis booigenitalium bovis canis capricolum columbinum equigenitalium felis hyorhinis (BTS7) M. hyorhinis M. ovipneumoniae M. pulmonis M. testudinis Anaerobe M. sp.a Anaerobe M. sp.b Anaerobe M. sp.b A. equifetale A. hippikon A. laidlawii U. sp.a U. sp.b Erysipelothrix rhusiopathiae
ntg 0 0 nt nt Traces 0 nt Traces
nt 10.1 i 2.1 8.3 + 1.9 nt nt 15.4 + 2.3 9.7 + 2.1 nt 17.2 t 3.1
0 18.4 11.7 20.8 0 23.4 0 0 35.7
f0 f 1.4 * 1.0 k 2.1 f 0.2 rk 1.9 k 0.2 *o +- 2.4
18.0 26.7 25.8 22.4 17.0 27.0 25.3 33.4 35.1
f + f k f + 3t f +
0.9 1.3 1.3 1.2 0.8 1.4 1.2 1.7 1.7
nt 31.9 _t 9.3 0 kO.2 0 f0.2 nt 0 f0 nt 0 I?0 0 +o
nt nt nt Traces 0 0 0 0 nt nt Traces nt nt
nt nt nt 14.8 f 9.4 + 12.5 ? 10.1 * 9.3 f nt nt 25.4 k nt nt
19.0 15.0 0 13.8 0 27.0 25.9 17.1 42.0 18.6 14.8 14.6 13.0
+- 1.1 * 0.9 k 0.4 i 1.0 fO.l f 1.7 f 1.7 + 1.3 k 2.2 + 1.2 k 0.9 + 1.0 f 0.9
22.0 21.4 13.8 23.1 24.9 28.7 26.2 24.8 40.1 23.6 36.8 14.3 17.2
* f f ? zk + k + + + + + +
1.1 1.1 0.8 1.2 1.3 1.6 1.5 1.4 1.9 1.3 1.8 0.9 1.0
nt nt nt 1.4 i 0.5 nt 0 2k 0.0 11.7 f 2.7 0 k 0.0 14.4 f 1.1 15.2 + 0.7 8.4 I? 1.5 nt nt
19.7 + 1.5
22.9 If- 1.7
M. M. M. M. M. M. M. M. M. M.
6.2 k 1.3
Old
1.9 2.3 1.4 1.7 2.7
2.9
14.6 k 1.9
12.1 f 1.2
Heated’
Dialyzedd
Note. The mean values and standard deviations of six different determinations are presented. a SOD was determined with cytochrome c (units/mg) (27). * CAT was determined polarographically with Hz02 (nkat). ’ Freshly prepared cell lysates were used for the determination. d Freshly prepared cell lysates were dialyzed twice for 12 h against 1000 vol PBS at 4°C. e Old cell lysates which were stored between 1 and 3 years at -20°C were used for the determination. ‘Cell lysates (freshly prepared and old) in PBS were heated to 60°C for 30 s. g Not tested.
tions than SOD. Within two different cultures of the anaerobic mycoplasma strain sp. b, one culture exhibited CAT activity comparable to that of aerobic mycoplasmas, whereas in the other no catalase activity was detectable. Peroxidase activity was not detectable with either odianisidine or scopoletin. The sensitivity of the scopoletin test was about 1 rig/liter horseradish peroxidase (29). Ten mollicute species, M. arthritidis, M. bovigenitalium, M. capricolum, M. columbinum, M. felis, M. pulmonis, M, testudinis, A. laidlawii, and the anaerobic mycoplasmas sp. a and sp. b were tested for 0; production with lucigenin and for HzOz production with luminol (33) in PBS supplemented with 25 mmol/liter glucose at 37°C (Fig. 2). Light emission was prevented by the addition of SOD (1 pmollliter) and CAT (10 pmol/liter). Without glucose as substrate, no production of 0, and HzOz was detectable. The anaerobic mycoplasmas showed a significantly higher production of reactive oxy-
gen species than the aerobic species. While the production of reactive oxygen compounds by the aerobic mollicutes decreased after some minutes, the anaerobic strains liberated continuously reactive oxygen products for up to 1 h. Equivalent results were obtained in a quantitative assay using acetylated cytochrome c for determination of 0; (32) and scopoletin for determination of the HzOz production (29). Both reactions were prevented by the addition of SOD (1 pmol/liter) or CAT (10 pmol/ liter). Additionally, small amounts of 0; and Hz02 production were detected in M. pulmonis. No production of reactive oxygen species by M. capricolum, M. columbinum, and M. felis was observed with glucose as substrate. The primary radical produced was 0, as demonstrated by ESR spin trapping (34) (Fig. 3). The mollicutes showed decreasing amplitudes of the DMPO-OOH adduct from the anaerobic M. sp. a, to M. sp. b, M. testudinis, which is identical to A. laidlawii, M. bovigenetalium, and M. arthritidis, whereas the hyperfine split-
SUPEROXIDE
4
DISMUTASE
5
6
IN
8
7
77
MOLLICUTES
9
lo
II
12
13
-
FIG. 1. SOD activity staining for several mollicutes. Activity staining for SOD was performed on 7.5% a&amide gels with nitroblue tetrazolium-riboflavin (31). Cell lysates (100 ~1) of the following bacteria were separated: 1, Erys$elothrix rhusiopathiue-aerobic control organism (5.76 mg protein/ml); 2, M. equigenitalium (2.5 mg protein/ml); 3, M. hyorhinis (2.65 mg protein/ml); 4, M. pulmonis (8.5 mg protein/ml); 5, M. bouigenitalium (3.8 mg protein/ml); 6, A. laidlawii (4.6 mg protein/ml); 7, M. hyorhinis (1.2 mg protein/ml); 8, M. felis (3.0 mg protein/ml); 9, M. canis (0.8 mg protein/ml); 10, M. bouis (1.8 mg protein/ml); 11, M. anaerobe sp. b (2.1 mg protein/ml); 12, M. anaerobe sp. a (7.2 mg protein/ml); 13, M. arthritidis (2.3 mg protein/ml).
ting of the radical adduct was not altered. The addition of 1 pmol/liter Cu-Zn-SOD prevented the formation of the radical adducts. DISCUSSION
The present investigations indicate that mollicutes, including some strains of mycoplasmas and ureaplas-
mas, contain SODS, probably with Mn in the active center. In nondialyzed cell extracts no SOD activities or only traces were detectable, and activities were detectable only after dialysis of the cell lysates. This is a wellknown phenomenon. Several organisms were first described as lacking SOD, but later SOD was demonstrated in these organ-
I
6
0
10
20
30 time (mini
40
50
60
i
0
b
2
FIG. 2. Determination of the 0; and H202 production by several mollicutes. (a) Time course nescence with lucigenine (33): 1, M. anaerobe sp. a, 2. M. anaerobe sp. b, 3. M. testudinis = A. (b) Time course of the HzOz production determined by chemiluminescence with luminol(33): bouigenitalium, 4, A. laidlawii, 5. M. testudinis, 6, M. arthritidis. Samples were incubated with at 37°C.
4
6 time hid
8
10
12
of the 0, production determined by chemilumiluidlawii, 4, M. bouigenitulium, 5, M. arthritidis. 1, M. anaerobe sp. a, 2, M. anaerobe sp. b, 3, M. the reagents in PBS with 25 mmol/liter glucose
78
MEIER
AND
isms too (36-41). An increase in total SOD activity compared to that of the cell-free extracts after some preliminary purification steps was also observed in some cases (41-46). SOD activity is often not detectable in bacterial cell-free extracts, especially in those of anaerobes (unpublished data of this laboratory) and has been ascribed to a production of 0, by membrane fragments still present in the bacterial lysates, thus artificially minimizing the real SOD activity (46, 47). We assume that analogous enzymatic mechanisms interfering with the test systems caused the failure to detect SOD in the cell lysates of the mollicutes. By dialyzing, the lysates were liberated from low-molecular-weight substrates for O;producing enzymes. In fact 0; production was observed in lysates of M. pneumoniue (6). Both membranes and cytoplasma caused an SOD inhibital reduction of cytochrome c attributed to a NAD(P)H-flavin oxidase. The presence of SOD in mycoplasmas might be heterogeneous, as in lactic acid bacteria and strains of Nelsseria gonorrhoeae. In crude extracts of A. laidlawii only traces of SOD activity were detectable, whereas in dialyzed and heat-treated extracts the activity was comparable to the results described by Lynch and Cole (7). Three of the thirteen strains tested for CAT activities were assayed previously by Lynch and Cole (7), who demonstrated the absence of CAT. We detected low CAT activities in M. arthritidis, M. pulmonis and A. laidZuwii by following the liberation of O2 from Hz02 polarographically. Like this group, we could not detect any CAT activity by determining the change of HzOz absorption at 240 nm. Additionally, we demonstrated CAT activity for three other strains tested, while in seven other strains tested no CAT activities were detectable.
TABLE Determination
II
of 0; and Hz02 Production
Species M. arthritidis M. bovigenitalum M. capricolum M. columbinum M. felis M. pulmonis M. testudinis Anaerobe M. sp.a Anaerobe M. sp.b A. laidluwii
0,” 1,860 3,733 19 3 0 261 5,740 18,110 10,030 5,268
by Mollicutes HzOz *
f f * zk + !I + * + +
15.6 47.3 0.5 0.0 0.0 13.0 43.0 67.0 59.0 23.0
283 f 930 f 0 + 0 f 0 f 39+ 1185 -t 3750 2 2363 t 1317 r
9.0 22.1 0.0 0.0 0.0 1.2 18.3 51.0 69.0 34.0
Note. The mean values and standard deviations of six different determinations are presented. a 0, production was determined photometrically .at 550 nm by the reduction of acetylated cytochrome c (32) over a period of 30 min at 37°C (nmol/30 min x mg protein). *HZ02 production was determined fluorometrically with scopoletin (excitation 381 nm, emission 436 nm) (29) over a period of 30 min at room temperature, 22°C (nmol/30 min X mg protein).
HABERMEHL
t g =2.0047
ImT FIG. 3.
Radical production by M. anaerobe sp. a was determined by ESR-spin trapping. (a) M. anaerobe sp. a, with a protein concentration of 1 mg/ml, was incubated for 15 min with DMPO at room temperature, 22°C (34). Measuring conditions are described under Materials and Methods. (b) M. anaerobe sp. a was incubated in the presence of 1 pmollliter Cu-Zn-SOD as described above.
In a comparison of these results, it should be always kept in mind that the induction of CAT is usually much more dependent on the culture conditions than that of SOD. Therefore, within two different cultures of an anaerobic mycoplasma strain, CAT was lacking in one and present in the other. Reports on the presence of CAT activity in M. pneumoniae, a human pathogenic species, differ, too. While most reports described a lack of catalase in M. pneumoniae (7, 11, 16, 49) in some cases low CAT activities were determined (8,50-52). Although a weak correlation between CAT activity and the production of reactive oxygen compounds was observed, there were exceptions, the anaerobic mycoplasma strains sp. a and sp. b. While the production of 0, and Hz02 was more than twice the amounts liberated by aerobic mollicutes, the strains either lacked CAT activity or the activity was comparable to that of the aerobic mollicutes. SOD activities were also comparable to those in aerobic mollicutes. It might be speculated that “anaerobiosis” is due to an immoderate liberation of reactive oxygen compounds causing a “relative” deficiency of protective enzymes. With regard to the mollicutes as mostly aerobic organisms, the detection of SOD supports the theory that SOD acts as an obligate antioxidative enzyme. A correlation between the amounts of liberated reactive oxygen compounds and pathogenicity was not observed. The two anaerobic mycoplasma strains A. laidlawii and M. testudinis (Tables I and II) are not pathogenic but liberate 0, and H202. However, it is remarkable that those strains which failed to liberate reactive oxygen compounds were apathogenic. H202 might be one of the pathogenic factors, but the conclusion that an HzOz-producing organism should be pathogenic or that pathogenic organisms should liberate H202 is certainly incorrect. ACKNOWLEDGMENTS This work Ha 457/38-l.
was supported by Deutsche Forschungsgemeinschaft AZ. We thank Professor H. Kirchhoff for kindly providing
SUPEROXIDE
DISMUTASE
the mollicutes, Mrs. R. Schmidt for culturing these organisms, Dipl. Biol. M. Runge for culturing M. arthritidis, and Professor K. Petzold for kindly providing Erysipelothrix rhusiopathiae. Moreover, we gratefully appreciate the opportunities to perform chemiluminescence at the laboratories of Professor W. Leibold and ESR spectroscopy at the University of Bremen.
(Gilbert,
145, 442-
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