JouRNAL OF BACTzRiOLOGY, May 1976, p. 733-738 Copyright ©) 1976 American Society for Microbiology
Vol. 126, No. 2 Printed in U.S.A.
Product Analysis of Bisulfite Reductase Activity Isolated from Desulfovibrio vulgaris H. L. DRAKE AND J. M. AKAGI* Department of Microbiology, University of Kansas, Lawrence, Kansas
66045
Received for publication 12 December 1975
Bisulfite reductase was purified from extracts of Desulfovibrio vulgaris. By colorimetric analyses trithionate was found to be the major product, being formed in quantities 5 to 10 times more than two other detectable products, thiosulfate and sulfide. When [35 ]bisulfite was used as the substrate, all three products were radioactively labeled. Degradation of [35Sltrithionate showed that all of its sulfur atoms were equally labeled. In contrast, [35S]thiosulfate contained virtually all of the radioactivity in the sulfonate atom while the sulfane atom was unlabeled. These results, in conjunction with the finding that the sulfide was radioactive, led to the conclusion that bisulfite reductase reduced bisulfite to trithionate as the major product and sulfide as the minor product; the reason for the unusual labeling pattern found in the thiosulfate molecule was not apparent at this time. When bisulfite reductase was incubated with [35S]bisulfite in the presence of another protein fraction, FII, the thiosulfate formed from this reaction contained both sulfur atoms having equal radioactivity. This discovery, plus the fact that trithionate was not reduced to thiosulfate under identical conditions, led to the speculation that bisulfite could be reduced to thiosulfate by another pathway not involving trithionate.
The reduction of bisulfite to trithionate by a bisulfite reductase from Desulfovibrio gigas was first demonstrated by Lee and Peck (12). Kobayashi et al. (10) reported that the bisulfite reductase isolated from Desulfovibrio vulgaris formed thiosulfate and sulfide in addition to trithionate. Subsequently, Kobayashi et al. (8) and Jones and Skyring (6) demonstrated that the amount of each of the end products was dependent upon the conditions and methods of assay used in measuring bisulfite reduction. Parameters such as concentrations of hydrogenase, methyl viologen, substrate, and hydrogen ion were important factors in determining the major end products of the reaction. From 35S-labeling studies, Akagi et al. (2) proposed that the bisulfite reductase (P582), isolated from Desulfotomaculum nigrificans, reduced bisulfite to trithionate and that thiosulfate and sulfide were endogenous side products of the reaction. Chambers and Trudinger (3) concluded from their 35S-labeling studies on Desulfovibrio desulfuricans that thiosulfate and trithionate may not be intermediates in the dissimilatory sulfate-reducing process. This study was initiated to determine the products of enzymatic bisulfite reduction using 35S-labeled substrate. The possible relationship between this enzyme and the "thiosulfate-forming" system (18) was also investigated to clarify the bisulfite reduction process.
MATERIALS AND METHODS Organism. D. vulgaris NC1B 8303 was grown and harvested as previously described (1). Enzyme assay conditions. Bisulfite reductase activity was measured in Warburg flasks of 8-ml capacity using standard manometric techniques. The particulate hydrogenase of D. vulgaris was prepared as described earlier (18). Unless otherwise indicated, the standard assay mixture contained, in micromoles: potassium phosphate buffer, pH 6.0, 100; methyl viologen, 1.0; sodium bisulfite, 10; hydrogenase, 0.25 mg; and enzyme in a total volume of 1.1 ml. The center well contained 0.1 ml of 20% CdCl2 absorbed on fluted filter paper. The gas phase was H2, and the temperature was 30 C. Purification of bisulfite reductase. To avoid any confusion pertaining to the term bisulfite reductase, this enzyme is also known as desulfoviridin. In an earlier report (18) we stated that a thiosulfate-forming system from D. vulgaris consisted of two proteins, one of which was designated fraction III. This fraction exhibited spectral properties identical to desulfoviridin and was subsequently found to be the same protein (unpublished data). Therefore, fraction III (18) and desulfoviridin are synonymous with the term bisulfite reductase in this communication. The bisulfite reductase was purified by a modification of the method described for fraction III (18). After sucrose density centrifugation, the fraction III layer was collected and dialyzed against several changes of distilled water at 4 C. The dialyzed fraction was applied to a diethylaminoethyl-cellulose column (2 by 10 cm) (chloride). The green band, adsorbed at the top of the column, was washed con733
734
DRAKE AND AKAGI
secutively with 50 ml of 0.05 M potassium phosphate buffer, pH 7.6, 500 ml of 0.1 M phosphate buffer, pH 7.6, and 50 ml of 0.15 M phosphate buffer, pH 7.6. These washings were monitored at 280 nm for protein and were necessary to remove the minor proteins contaminating bisulfite reductase. The green pigment was eluted from the column with 0.3 M phosphate buffer, pH 7.6, and dialyzed against 100 volumes of distilled water for 24 h at 4 C. The enzyme was concentrated by pressure filtration using an Amicon EC-20 unit with a PM 30 filter. The major and minor bands observed when this bisulfite reductase was analyzed by polyacrylamide gel electrophoresis have been reported (5, 8, 12) to be characteristic for pure desulfoviridin. The other component of the thiosulfate-forming system, fraction II (FII), was obtained as described previously (18). Analytical determinations. Thiosulfate and trithionate analyses were performed according to Kelly et al. (7) as described previously (2). Sulfide was determined by analyzing the filter paper in the center well by the method of Fogo and Popowski (4). Radioactive sulfide was analyzed as described earlier (2). Protein was estimated by the method of Lowry et al. (14), using bovine serum albumin as a standard. Isolation of 35S-labeled trithionate and thiosulfate from reaction mixtures was accomplished by thin-layer chromatography, described earlier (2). Trithionate was degraded as follows. After scraping the area on the silica gel corresponding to known trithionate, 10 to 15 ml of water was added to elute the compound. To a suitable fraction of the eluate was added 1 ml of 0.25 M KCN, and the mixture was made alkaline with a few drops of 2 N NaOH. The solution was placed in a boiling-water bath for 1 h and cooled to room temperature. It was acidified by dropwise addition of concentrated H2SO4, and 0.2 M AgNO3 was added until no more precipitation was visible. The reactions occurring are:
03S - S* - S032 - + 3CN- + H20 -3 SO32 - + S*CN+ S042-- + 2HCN Ag NO3 + S*CN- -* Ag S*CN + NO3The AgSCN precipitate was collected by filtration on Whatman no. 42 paper, washed with water, dried, and counted. The filtrate, containing the two outer sulfonate atoms (SO32- and SO42), was treated with excess NaCl to remove residual silver ions, and the AgCl precipitate was removed by filtration. The filtrate from this step was treated with 0.1 N I2 solution (dropwise) until a straw-colored filtrate was obtained. This step oxidized whatever S032- was present to SO42-. A saturated solution of BaCl2 was added to precipitate the BaSO4, and this was collected by filtration. The BaSO4 precipitate, representing the two outer sulfur atoms, was collected by filtration and dried. Its radioactivity was determined by placing the filter paper into scintillation vials. Thiosulfate was isolated by the thin-layer chromatography procedure described above. The region corresponding to known thiosulfate was scraped off the plate and stirred with distilled water
J . BACTERIOL .
for 30 min to elute the compound. The eluate, supplemented with 10 ,tmol of Na2S2O3, was applied to a Dowex-1 column (7 by 150 mm, nitrate form, 100 to 200 mesh), and the column was washed with 0.1 M NaNO3 to remove any contaminating SO32- and S042-. The 0.1 M eluate was monitored for 35 O4 by periodically checking for radioactivity and by its ability to form a precipitate with BaC12. Thiosulfate was removed from the column with 1 M NaNO3 (17). A suitable portion of the thiosulfate-containing fraction was degraded by making the solution alkaline with 1 N NaOH and adding 1 ml of 0.25 M KCN. After 10 min at room temperature, 1.5 ml of 0.2 M AgNO3 was added to precipitate the sulfane atom as AgSCN. The precipitate was collected by filtration, washed with water, dried, and counted. The filtrate containing the sulfonate atom was treated with NaCl and I2, as described above for trithionate degradation, followed by BaCl2 to precipitate the BaSO4 (sulfonate sulfur). This was counted in a scintillation vial. Thiosulfate was also degraded as described previously (2), and both methods gave similar results. In addition, authentic sulfane and sulfonatelabeled [355]thiosulfate were degraded using the methods described to confirm the accuracy of the degradation methods. Sodium bisulfite solutions were prepared fresh for each experiment in 0.001 M disodium ethylenediaminetetraacetate. Periodically the Na235SO3 solutions were checked for any oxidation to Na235SO4 by acid volatilization and trapping the volatile 35SO2 in hyamine hydroxide. Sodium [35S]sulfite was purchased from New England Nuclear Corp. Sulfane and sulfonate-labeled [35S]thiosulfite were obtained from Amersham/Searle Corp. Liquid scintillation counting was performed with a Beckman LS-100C and a Packard Tri-Carb, model 3375, spectrometer. All counts were corrected for quench and background. Precoated preparative thin-layer chromatography plates (silica G-200), were purchased from Brinkmann Instruments, Inc. Trithionate was synthesized as described previously (2).
RESULTS General properties of enzymes. Bisulfite reductase solutions were stable for several months at -20 C, whereas repeated thawing and freezing resulted in a slow progressive loss of activity. For this reason the enzyme was stored frozen in 1-ml aliquots and thawed once for each experiment. The optimum pH for activity was 6.0. The absorption spectrum for bisulfite reductase exhibited peak maxima at 630, 583, 410, 390, and 280 nm. Trithionate and thiosulfate were not reduced by bisulfite reductase. Fraction II, the other component of the thiosulfate-forming system, (18) contained approximately 10 different proteins as judged by polyacrylamide gel electrophoresis. It was used in this study without any further purification. This fraction was stable to lyophilization and was stored at -20 C in the dried state. Desul-
VOL. 126, 1976
foviridin was absent in the FII preparations as judged by the alkaline fluorescent diagnostic test for this pigment (16). Products of bisulfite reductase activity. When bisulfite reductase was incubated with bisulfite under standard assay conditions, the products detected were trithionate as the predominant species, with thiosulfate and sulfide appearing in lesser amounts. This is in agreement with results obtained by other investigators (5, 8, 10) and with those reported for the bisulfite reductase (P582) activity isolated from D. nigrificans (2). Effect of time on products formation. Figure 1 shows the formation of three products of bisulfite reduction as a function of time. Trithionate is formed at a rate significantly faster than thiosulfate and sulfide. Effect of enzyme concentration on bisulfite reduction. Figure 2 shows the effect of enzyme concentration on bisulfite reduction products. Trithionate is seen to be the major product, and its formation correlates well with increasing enzyme concentration. Thiosulfate and sulfide may be the result of non-enzymatic reactions. Degradation of [35S]trithionate and
[35SIthiosulfate formed by bisulfite reductase. Bisulfite reductase was incubated with H35SO3-under standard assay conditions and the 35S_ labeled products, trithionate and thiosulfate, were isolated by thin-layer and/or column chromatography. Table 1 shows the distribution of 35S in trithionate. It is seen that all three sulfur atoms were equivalently radioactive, indicating that they all arose from the substrate H35SO-. In contrast, Table 2 shows that the sulfur atoms comprising the thiosulfate molecule were not equally labeled. The sulfonate
33
-o./ E
01 UP
L
0 -
0
TABLE 1. Degradation of 35S-labeled trithionate isolated from reaction mixturesa Expt
Trithionate (counts/min) Counts/ min deS 03S S03 graded
%
Reev-
eov
ery 81 61 93
7,672 14,005 10,770
1,864 2,482 1,864 2,510 3,541 2,510 3,953 2,130 3,953 Avg dis- 33.5 33 33.5 tribution
1 2 3
(%M_
Enzyme concentration, 0.35 mg; Hn5SO3- concentration, 10 mM containing 105 counts/min per ,umol; incubation time, 90 min. a
TABLE 2. Distribution of 35S in thiosulfate isolated from.reaction mixturesa Thiosulfate Counts/
Avg distribution
(1)
150
FIG. 2. Effect of bisulfite reductase concentration on bisulfite reduction products formation. Standard assay mixture. Incubation time, 90 min. Symbols: Trithionate, 0; thiosulfate, 0; sulfide, A.
1 2 3 (
0
120 90 60 TIME (minutes)
30
Expt
~1: C2 w a:
%
(counts/min) S S03 2,216 2,369 2,554
10,192 18,674 14,428
13.7
86.3
_
_
_
_
min de-
% Recovery
graded
20,938 19,860 16,550 _
_
_
59 106 103 _
_
_
Enzyme concentration, 0.35 mg; H35SO3- concentration, 20 mM containing 105 counts/min per I
HnX. 0.25 0.50 PROTEIN (mg) FIG. 1. Effect of time on products formation. Standard assay mixture. Bisulfite reductase, 0.35 mg. Symbols: Trithionate, 0; thiosulfate, 0; sulfide, A.
735
BISULFITE REDUCTASE OF D. VULGARIS
,umol; incubation time, 90 min. sulfur contained most of the radioactivity, whereas the sulfane atom was slightly radioactive. This labeling pattern was previously observed by Levinthal and Schiff (13) with cell extracts of Chlorella pyrenoidosa and by Akagi
736
J. BACTERIOL.
DRAKE AND AKAGI
et al. (2) with the bisulfite reductase (P582)
isolated from D. nigrificans (2). Formation of [35S]sulfide by bisulfite reductase. The reduction of H35S03-- to 35S2-- by bifulfite reductase from D. vulgaris was observed by Kobayashi et al. (8), whereas P582 from D. nigrificans did not catalyze this reaction (2). In this study we consistently observed 35S2- formation from H35S03-- and thus concur with the results of Kobayashi et al. (8). However, the amount of radioactivity detected in the sulfide was consistent with the amount estimated by the colorimetric method (Fig. 1 and 2). Incubation of 0.35 mg of bisulfite reductase with H35S03-- (20 gmol containing 105 counts/min per ,mol) for 90 min resulted in 0.3 gmol of 35S2-- containing 35,000 counts/min, whereas the radioactivity in the trithionate fraction was 106 counts/min. Since there were approximately 2 ,u mol of trithionate formed during this time, the major pathway of bisulfite reduction by bisulfite reductase appears to be in the direction of trithionate formation. Conditions for increased thiosulfate formation. Suh and Akagi (18) reported that a thiosulfate-forming system consisted of two fractions (FII and FIII). FIII was found to correspond to bisulfite reductase, whereas FII consisted of an unpurified protein fraction. Incubating FII and bisulfite reductase, either alone or in combination, did not reduce trithionate to thiosulfate. However, when both fractions were incubated with bisulfite, thiosulfate was formed in quantities greater than with bisulfite reductase alone (Table 3). The presence of FII increased trithionate accumulation, as well as that of thiosulfate, over that formed by bisulfite reductase alone. The ratio of trithionate to thiosulfate, in the presence of FII and bisulfite reductase, is considerably lower than the ratio produced by bisulfite reductase alone. If, in the presence of both fractions, bisulfite were reduced to thiosulfate through a pathway not involving trithionate, the sulfur atoms of thiosulfate, formed by this system, should have originated from bisulfite. Table 4 shows the distribution of radioactivity in the thi6sulfate molecule formed by FII plus bisulfite reductase. The amount of radioactivity in the sulfane atom approaches that found in the sulfonate atom. If we assume that the slight increase in radioactivity of the sulfonate sulfur atom is due to the contribution of thiosulfate formation by bisulfite reductase alone, as shown in Table 2, then the data shown in Table 4 indicate that FII plus bisulfite reductase form thiosulfate by a mechanism different than that used by bisulfite reductase alone.
TABLE 3. Bisulfite reduction products formed by the thiosulfate-forming system
Amol System
S3062-
S20321.54 0.09 0.49
S2-
0.21 2.89 Completea 0.19 1.08 Minus FIH 0.38 0.17 Minus bisulfite reductase 0.18 0.22 1.72 Minus FII + boiled FlIb a Complete system consisted of the standard assay mixture plus FII. Bisulfite reductase concentration, 0.42 mg; FII, 1.4 mg. bFII was placed in a boiling-water bath for 5 min. TABLE 4. Degradation of [35S]thiosulfate formed by the thiosulfate-forming system' Counts/ Thiosulfate (counts/min) min de- % Recovery Expt graded S SO, 101 25,129 38,025 62,500 1 81 29,392 31,459 74,600 2 94 5,220 7,155 13,200 3 56 44 Avg distri-
bution a Reaction mixture was the same as that described for the complete system in Table 3. Bisulfite reductase concentration, 0.42 mg; FII, 1.4 mg; incubation time, 90 min.
DISCUSSION The results obtained in this study indicate that the bisulfite reductase, isolated from D.
vulgaris, reduces bisulfite to trithionate as the
major product and to sulfide as a minor product. Thiosulfate is not considered to be a product of the enzymatic process according to the observed labeling pattern. It is possible that the sulfane atom of this molecule originated from the proteins present in the reaction mixture. This explanation was given for the identical labeling pattern found in thiosulfate isolated from reaction mixtures containing C. pyrenoi-
dosa (13) and bisulfite reductase from D. nigriformed by D. vulgaris bisulfite reductase, it appears that this enzyme differs from its counterpart in D. nigrificans, which, reportedly,
ficans (2). Because small amounts of 3S2- are
does not reduce HMS03S2-- to 35S2--. Since it was previously demonstrated (6, 8) that the bisulfite reductase (desulfoviridin) from D. gigas and D. vulgaris formed trithionate and sulfide in varying amounts depending upon the assay condi-
tions, it is possible that the D. nigrificans bisulfite reductase (P582) can also reduce bisulfite
VOL. 126, 1976
BISULFITE REDUCTASE OF D. VULGARIS
to sulfide. Perhaps the conditions used in the studies by Akagi et al. (2) were unfavorable for sulfide formation. It can also be speculated that the desulfoviridin molecule contains an active site that can slowly reduce the intermediate species, between bisulfite and trithionate, to sulfide and that this property is not inherent to P582. Whatever the reason may be for this difference between the two bisulfite reductases, we believe that the main role for these enzymes is to form trithionate from bisulfite. Chambers and Trudinger (3) questioned the possibility that trithionate and thiosulfate are intermediates in dissimilatory sulfate reduction. Their conclusions were mainly based on studies involving washed and growing cells of D. desulfuricans and D. gigas. Whereas whole cell experiments can yield valuable information, there can occasionally be equivocal data obtained from such studies. Whole cells of D. desulfuricans and D. gigas were observed to reduce both sulfur atoms of thiosulfate at relatively equal rates (3). In addition, when 35SO42and unlabeled thiosulfate were incubated with D. desulfuricans cells the specific activity of the accumulated 35S2- was found not to be identical to that of the sulfane atom of thiosulfate. These data led Chambers and Trudinger (3) to conclude that thiosulfate was not an intermediate in the sulfate-reducing prpcess. Nakatsukasa and Akagi (15) observed that a partially purified thiosulfate reductase from D. nigrificans reduced both sulfur atoms of thiosulfate to sulfide at equal rates; however, a further purification of this enzyme showed that it reduced only the sulfane atom ofthiosulfate. It is, therefore, not surprising that whole cells can reduce the sulfane and sulfonate sulfur atoms to sulfide at equal rates. Furthermore, if two different substrates, such as sulfate and thiosulfate, are simultaneously incubated with whole cells to compare their rates of metabolism, it should be established that their rates of uptake are equal. On the basis of their conclusion that thiosulfate is not an intermediate in the sulfate reduction process, and since they observed that trithionate is reduced to thiosulfate, Chambers and Trudinger (3) suggested that trithionate is probably not an intermediate between bisulfite and sulfide. Because trithionate is a product of bisulfite reductase activity (5, 8-10, 12) and because we have evidence for the presence of a trithionate reductase activity in sulfate-reducing bacteria (unpublished data), we feel that one pathway for bisulfite reduction to sulfide involves trithionate and thiosulfate, as proposed by Kabayashi et al. (9). A second path-
737
way for sulfite reduction is catalyzed by an assimilatory sulfite reductase present in these organisms (11). Prior to this study we assumed that thiosulfate formation by bisulfite reductase plus FII (thiosulfate-forming system) occurred by the sequential reduction of bisulfite to trithionate to thiosulfate. However, the observations that this mixture did not reduce trithionate and the 35S-labeling pattern shown in Table 4 indicated that thiosulfate was formed through a pathway not involving trithionate. We propose that another pathway for bisulfite reduction, which involves the thiosulfate-forming system (18), is possible in D. vulgaris. We are currently attempting to purify the active protein of this system from the FII fraction. ACKNOWLEDGMENTS This study was supported in part by a grant, GB-37545, from the National Science Foundation, by Public Health Service grant AI-04672 from the National Institute of Allergy and Infectious Diseases, by Public Health Service training grant GM-703 from the National Institute of General Medical Sciences, and by grants from the University of Kansas Biomedical Sciences Support Committee and the General Research Fund. J. M. A. is a recipient of Research Career Development award GM-30,262 from the National Institute of General Medical Sciences.
LITERATURE CITED 1. Akagi, J. M., and L. L. Campbell. 1962. Studies on thermophilic sulfate-reducing bacteria. III. Adenosine triphosphate-sulfurylase of Clostridium nigrificans and Desulfovibrio desulfuricans. J. Bacteriol. 84:1194-1201. 2. Akagi, J. M., M. Chan, and V. Adams. 1974. Observations on the bisulfite reductase (P582) isolated from Desulfotomaculum nigrificans. J. Bacteriol. 120:240244. 3. Chambers, L. A., and P. A. Trudinger. 1975. Are thiosulfate and trithionate intermediates in dissimilatory sulfate reduction? J. Bacteriol. 123:36-40. 4. Fogo, J. K., and M. Popowski. 1949. Spectrophotometric determination of hydrogen sulfide. Anal. Chem. 21:732-734. 5. Jones, H. E., and G. W. Skyring. 1974. Reduction of sulfite to sulfide catalyzed by desulfoviridin from De-
sulfovibrio gigas. Aust. J. Biol. Sci. 27:7-14. 6. Jones, H. E., and G. W. Skyring. 1975. Effect of enzymic assay conditions on sulfite reduction catalyzed by desulfoviridin from Desulfovibrio gigas. Biochim. Biophys. Acta 377:52-60. 7. Kelly, D. P., L. A. Chambers, and P. A. Trudinger. 1969. Cyanolysis and spectrophotometric estimation of trithionate in mixture with thiosulfate and tetrathionate. Anal. Chem. 41:898-901. 8. Kobayashi, K., Y. Seki, and M. Ishimoto. 1974. Biochemical studies on sulfate reducing bacteria. XIII. Sulfite reductase from Desulfovibrio vulgaris. Mechanism of trithionate, thiosulfate, and sulfide formation and enzymatic properties. J. Biochem. (Tokyo) 75:519-529. 9. Kobayashi, K., S. Tachibana, and M. Ishimoto. 1969. Intermediary formation of trithionate in sulfite reduction by a sulfate-reducing bacterium. J. Biochem. (Tokyo) 65:155-157.
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10. Kobayashi, K., E. Takahashi, and M. Ishimoto. 1972. Biochemical studies on sulfate-reducing bacteria. XI. Purification and some properties of sulfite reductase, desulfoviridin. J. Biochem. (Tokyo) 72:879-887. 11. Lee, J. P., J. LeGalland, and H. D. Peck. 1973. Isolation of assimilatory and dissimilatory-type sulfite reductases from Desulfovibrio vulgaris. J. Bacteriol. 115:529-542. 12. Lee, J. P., and H. D. Peck. 1971. Purification of the enzyme reducing bisulfite to trithionate from Desulfovibrio gigas and its identification as desulfoviridin.
Biochem. Biophys. Res. Commun. 45:583-589. 13. Levinthal, M., and J. A. Schiff. 1968. Studies on sulfate utilization by algae. 5. Identification of thiosulfate as the major acid-volatile product formed by a cell-free sulfate-reducing system from Chlorella. Plant Phys-
iol. 43:555-562. 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Nakatsukasa, W., and J. M. Akagi. 1969. Thiosulfate reductase isolated from Desulfotomaculum nigrificans. J. Bacteriol. 98:429-433. Postgate, J. R. 1959. A diagnostic reaction of Desulphovibrio desulphuricans. Nature (London) 183:481482. Samuelson, 0. 1963. Ion exchange separation in analytical chemistry, p. 416-418. John Wiley and Sons, Inc., New York. Suh, B., and J. M. Akagi. 1969. Formation of thiosulfate from sulfite by Desulfovibrio vulgaris. J. Bacteriol. 99:210-215.
14. Lowry,
15.
16. 17.
18.
Vol. 126, No. 2 Printed in U.S.A.
Product Analysis of Bisulfite Reductase Activity Isolated from Desulfovibrio vulgaris H. L. DRAKE AND J. M. AKAGI* Department of Microbiology, University of Kansas, Lawrence, Kansas
66045
Received for publication 12 December 1975
Bisulfite reductase was purified from extracts of Desulfovibrio vulgaris. By colorimetric analyses trithionate was found to be the major product, being formed in quantities 5 to 10 times more than two other detectable products, thiosulfate and sulfide. When [35 ]bisulfite was used as the substrate, all three products were radioactively labeled. Degradation of [35Sltrithionate showed that all of its sulfur atoms were equally labeled. In contrast, [35S]thiosulfate contained virtually all of the radioactivity in the sulfonate atom while the sulfane atom was unlabeled. These results, in conjunction with the finding that the sulfide was radioactive, led to the conclusion that bisulfite reductase reduced bisulfite to trithionate as the major product and sulfide as the minor product; the reason for the unusual labeling pattern found in the thiosulfate molecule was not apparent at this time. When bisulfite reductase was incubated with [35S]bisulfite in the presence of another protein fraction, FII, the thiosulfate formed from this reaction contained both sulfur atoms having equal radioactivity. This discovery, plus the fact that trithionate was not reduced to thiosulfate under identical conditions, led to the speculation that bisulfite could be reduced to thiosulfate by another pathway not involving trithionate.
The reduction of bisulfite to trithionate by a bisulfite reductase from Desulfovibrio gigas was first demonstrated by Lee and Peck (12). Kobayashi et al. (10) reported that the bisulfite reductase isolated from Desulfovibrio vulgaris formed thiosulfate and sulfide in addition to trithionate. Subsequently, Kobayashi et al. (8) and Jones and Skyring (6) demonstrated that the amount of each of the end products was dependent upon the conditions and methods of assay used in measuring bisulfite reduction. Parameters such as concentrations of hydrogenase, methyl viologen, substrate, and hydrogen ion were important factors in determining the major end products of the reaction. From 35S-labeling studies, Akagi et al. (2) proposed that the bisulfite reductase (P582), isolated from Desulfotomaculum nigrificans, reduced bisulfite to trithionate and that thiosulfate and sulfide were endogenous side products of the reaction. Chambers and Trudinger (3) concluded from their 35S-labeling studies on Desulfovibrio desulfuricans that thiosulfate and trithionate may not be intermediates in the dissimilatory sulfate-reducing process. This study was initiated to determine the products of enzymatic bisulfite reduction using 35S-labeled substrate. The possible relationship between this enzyme and the "thiosulfate-forming" system (18) was also investigated to clarify the bisulfite reduction process.
MATERIALS AND METHODS Organism. D. vulgaris NC1B 8303 was grown and harvested as previously described (1). Enzyme assay conditions. Bisulfite reductase activity was measured in Warburg flasks of 8-ml capacity using standard manometric techniques. The particulate hydrogenase of D. vulgaris was prepared as described earlier (18). Unless otherwise indicated, the standard assay mixture contained, in micromoles: potassium phosphate buffer, pH 6.0, 100; methyl viologen, 1.0; sodium bisulfite, 10; hydrogenase, 0.25 mg; and enzyme in a total volume of 1.1 ml. The center well contained 0.1 ml of 20% CdCl2 absorbed on fluted filter paper. The gas phase was H2, and the temperature was 30 C. Purification of bisulfite reductase. To avoid any confusion pertaining to the term bisulfite reductase, this enzyme is also known as desulfoviridin. In an earlier report (18) we stated that a thiosulfate-forming system from D. vulgaris consisted of two proteins, one of which was designated fraction III. This fraction exhibited spectral properties identical to desulfoviridin and was subsequently found to be the same protein (unpublished data). Therefore, fraction III (18) and desulfoviridin are synonymous with the term bisulfite reductase in this communication. The bisulfite reductase was purified by a modification of the method described for fraction III (18). After sucrose density centrifugation, the fraction III layer was collected and dialyzed against several changes of distilled water at 4 C. The dialyzed fraction was applied to a diethylaminoethyl-cellulose column (2 by 10 cm) (chloride). The green band, adsorbed at the top of the column, was washed con733
734
DRAKE AND AKAGI
secutively with 50 ml of 0.05 M potassium phosphate buffer, pH 7.6, 500 ml of 0.1 M phosphate buffer, pH 7.6, and 50 ml of 0.15 M phosphate buffer, pH 7.6. These washings were monitored at 280 nm for protein and were necessary to remove the minor proteins contaminating bisulfite reductase. The green pigment was eluted from the column with 0.3 M phosphate buffer, pH 7.6, and dialyzed against 100 volumes of distilled water for 24 h at 4 C. The enzyme was concentrated by pressure filtration using an Amicon EC-20 unit with a PM 30 filter. The major and minor bands observed when this bisulfite reductase was analyzed by polyacrylamide gel electrophoresis have been reported (5, 8, 12) to be characteristic for pure desulfoviridin. The other component of the thiosulfate-forming system, fraction II (FII), was obtained as described previously (18). Analytical determinations. Thiosulfate and trithionate analyses were performed according to Kelly et al. (7) as described previously (2). Sulfide was determined by analyzing the filter paper in the center well by the method of Fogo and Popowski (4). Radioactive sulfide was analyzed as described earlier (2). Protein was estimated by the method of Lowry et al. (14), using bovine serum albumin as a standard. Isolation of 35S-labeled trithionate and thiosulfate from reaction mixtures was accomplished by thin-layer chromatography, described earlier (2). Trithionate was degraded as follows. After scraping the area on the silica gel corresponding to known trithionate, 10 to 15 ml of water was added to elute the compound. To a suitable fraction of the eluate was added 1 ml of 0.25 M KCN, and the mixture was made alkaline with a few drops of 2 N NaOH. The solution was placed in a boiling-water bath for 1 h and cooled to room temperature. It was acidified by dropwise addition of concentrated H2SO4, and 0.2 M AgNO3 was added until no more precipitation was visible. The reactions occurring are:
03S - S* - S032 - + 3CN- + H20 -3 SO32 - + S*CN+ S042-- + 2HCN Ag NO3 + S*CN- -* Ag S*CN + NO3The AgSCN precipitate was collected by filtration on Whatman no. 42 paper, washed with water, dried, and counted. The filtrate, containing the two outer sulfonate atoms (SO32- and SO42), was treated with excess NaCl to remove residual silver ions, and the AgCl precipitate was removed by filtration. The filtrate from this step was treated with 0.1 N I2 solution (dropwise) until a straw-colored filtrate was obtained. This step oxidized whatever S032- was present to SO42-. A saturated solution of BaCl2 was added to precipitate the BaSO4, and this was collected by filtration. The BaSO4 precipitate, representing the two outer sulfur atoms, was collected by filtration and dried. Its radioactivity was determined by placing the filter paper into scintillation vials. Thiosulfate was isolated by the thin-layer chromatography procedure described above. The region corresponding to known thiosulfate was scraped off the plate and stirred with distilled water
J . BACTERIOL .
for 30 min to elute the compound. The eluate, supplemented with 10 ,tmol of Na2S2O3, was applied to a Dowex-1 column (7 by 150 mm, nitrate form, 100 to 200 mesh), and the column was washed with 0.1 M NaNO3 to remove any contaminating SO32- and S042-. The 0.1 M eluate was monitored for 35 O4 by periodically checking for radioactivity and by its ability to form a precipitate with BaC12. Thiosulfate was removed from the column with 1 M NaNO3 (17). A suitable portion of the thiosulfate-containing fraction was degraded by making the solution alkaline with 1 N NaOH and adding 1 ml of 0.25 M KCN. After 10 min at room temperature, 1.5 ml of 0.2 M AgNO3 was added to precipitate the sulfane atom as AgSCN. The precipitate was collected by filtration, washed with water, dried, and counted. The filtrate containing the sulfonate atom was treated with NaCl and I2, as described above for trithionate degradation, followed by BaCl2 to precipitate the BaSO4 (sulfonate sulfur). This was counted in a scintillation vial. Thiosulfate was also degraded as described previously (2), and both methods gave similar results. In addition, authentic sulfane and sulfonatelabeled [355]thiosulfate were degraded using the methods described to confirm the accuracy of the degradation methods. Sodium bisulfite solutions were prepared fresh for each experiment in 0.001 M disodium ethylenediaminetetraacetate. Periodically the Na235SO3 solutions were checked for any oxidation to Na235SO4 by acid volatilization and trapping the volatile 35SO2 in hyamine hydroxide. Sodium [35S]sulfite was purchased from New England Nuclear Corp. Sulfane and sulfonate-labeled [35S]thiosulfite were obtained from Amersham/Searle Corp. Liquid scintillation counting was performed with a Beckman LS-100C and a Packard Tri-Carb, model 3375, spectrometer. All counts were corrected for quench and background. Precoated preparative thin-layer chromatography plates (silica G-200), were purchased from Brinkmann Instruments, Inc. Trithionate was synthesized as described previously (2).
RESULTS General properties of enzymes. Bisulfite reductase solutions were stable for several months at -20 C, whereas repeated thawing and freezing resulted in a slow progressive loss of activity. For this reason the enzyme was stored frozen in 1-ml aliquots and thawed once for each experiment. The optimum pH for activity was 6.0. The absorption spectrum for bisulfite reductase exhibited peak maxima at 630, 583, 410, 390, and 280 nm. Trithionate and thiosulfate were not reduced by bisulfite reductase. Fraction II, the other component of the thiosulfate-forming system, (18) contained approximately 10 different proteins as judged by polyacrylamide gel electrophoresis. It was used in this study without any further purification. This fraction was stable to lyophilization and was stored at -20 C in the dried state. Desul-
VOL. 126, 1976
foviridin was absent in the FII preparations as judged by the alkaline fluorescent diagnostic test for this pigment (16). Products of bisulfite reductase activity. When bisulfite reductase was incubated with bisulfite under standard assay conditions, the products detected were trithionate as the predominant species, with thiosulfate and sulfide appearing in lesser amounts. This is in agreement with results obtained by other investigators (5, 8, 10) and with those reported for the bisulfite reductase (P582) activity isolated from D. nigrificans (2). Effect of time on products formation. Figure 1 shows the formation of three products of bisulfite reduction as a function of time. Trithionate is formed at a rate significantly faster than thiosulfate and sulfide. Effect of enzyme concentration on bisulfite reduction. Figure 2 shows the effect of enzyme concentration on bisulfite reduction products. Trithionate is seen to be the major product, and its formation correlates well with increasing enzyme concentration. Thiosulfate and sulfide may be the result of non-enzymatic reactions. Degradation of [35S]trithionate and
[35SIthiosulfate formed by bisulfite reductase. Bisulfite reductase was incubated with H35SO3-under standard assay conditions and the 35S_ labeled products, trithionate and thiosulfate, were isolated by thin-layer and/or column chromatography. Table 1 shows the distribution of 35S in trithionate. It is seen that all three sulfur atoms were equivalently radioactive, indicating that they all arose from the substrate H35SO-. In contrast, Table 2 shows that the sulfur atoms comprising the thiosulfate molecule were not equally labeled. The sulfonate
33
-o./ E
01 UP
L
0 -
0
TABLE 1. Degradation of 35S-labeled trithionate isolated from reaction mixturesa Expt
Trithionate (counts/min) Counts/ min deS 03S S03 graded
%
Reev-
eov
ery 81 61 93
7,672 14,005 10,770
1,864 2,482 1,864 2,510 3,541 2,510 3,953 2,130 3,953 Avg dis- 33.5 33 33.5 tribution
1 2 3
(%M_
Enzyme concentration, 0.35 mg; Hn5SO3- concentration, 10 mM containing 105 counts/min per ,umol; incubation time, 90 min. a
TABLE 2. Distribution of 35S in thiosulfate isolated from.reaction mixturesa Thiosulfate Counts/
Avg distribution
(1)
150
FIG. 2. Effect of bisulfite reductase concentration on bisulfite reduction products formation. Standard assay mixture. Incubation time, 90 min. Symbols: Trithionate, 0; thiosulfate, 0; sulfide, A.
1 2 3 (
0
120 90 60 TIME (minutes)
30
Expt
~1: C2 w a:
%
(counts/min) S S03 2,216 2,369 2,554
10,192 18,674 14,428
13.7
86.3
_
_
_
_
min de-
% Recovery
graded
20,938 19,860 16,550 _
_
_
59 106 103 _
_
_
Enzyme concentration, 0.35 mg; H35SO3- concentration, 20 mM containing 105 counts/min per I
HnX. 0.25 0.50 PROTEIN (mg) FIG. 1. Effect of time on products formation. Standard assay mixture. Bisulfite reductase, 0.35 mg. Symbols: Trithionate, 0; thiosulfate, 0; sulfide, A.
735
BISULFITE REDUCTASE OF D. VULGARIS
,umol; incubation time, 90 min. sulfur contained most of the radioactivity, whereas the sulfane atom was slightly radioactive. This labeling pattern was previously observed by Levinthal and Schiff (13) with cell extracts of Chlorella pyrenoidosa and by Akagi
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J. BACTERIOL.
DRAKE AND AKAGI
et al. (2) with the bisulfite reductase (P582)
isolated from D. nigrificans (2). Formation of [35S]sulfide by bisulfite reductase. The reduction of H35S03-- to 35S2-- by bifulfite reductase from D. vulgaris was observed by Kobayashi et al. (8), whereas P582 from D. nigrificans did not catalyze this reaction (2). In this study we consistently observed 35S2- formation from H35S03-- and thus concur with the results of Kobayashi et al. (8). However, the amount of radioactivity detected in the sulfide was consistent with the amount estimated by the colorimetric method (Fig. 1 and 2). Incubation of 0.35 mg of bisulfite reductase with H35S03-- (20 gmol containing 105 counts/min per ,mol) for 90 min resulted in 0.3 gmol of 35S2-- containing 35,000 counts/min, whereas the radioactivity in the trithionate fraction was 106 counts/min. Since there were approximately 2 ,u mol of trithionate formed during this time, the major pathway of bisulfite reduction by bisulfite reductase appears to be in the direction of trithionate formation. Conditions for increased thiosulfate formation. Suh and Akagi (18) reported that a thiosulfate-forming system consisted of two fractions (FII and FIII). FIII was found to correspond to bisulfite reductase, whereas FII consisted of an unpurified protein fraction. Incubating FII and bisulfite reductase, either alone or in combination, did not reduce trithionate to thiosulfate. However, when both fractions were incubated with bisulfite, thiosulfate was formed in quantities greater than with bisulfite reductase alone (Table 3). The presence of FII increased trithionate accumulation, as well as that of thiosulfate, over that formed by bisulfite reductase alone. The ratio of trithionate to thiosulfate, in the presence of FII and bisulfite reductase, is considerably lower than the ratio produced by bisulfite reductase alone. If, in the presence of both fractions, bisulfite were reduced to thiosulfate through a pathway not involving trithionate, the sulfur atoms of thiosulfate, formed by this system, should have originated from bisulfite. Table 4 shows the distribution of radioactivity in the thi6sulfate molecule formed by FII plus bisulfite reductase. The amount of radioactivity in the sulfane atom approaches that found in the sulfonate atom. If we assume that the slight increase in radioactivity of the sulfonate sulfur atom is due to the contribution of thiosulfate formation by bisulfite reductase alone, as shown in Table 2, then the data shown in Table 4 indicate that FII plus bisulfite reductase form thiosulfate by a mechanism different than that used by bisulfite reductase alone.
TABLE 3. Bisulfite reduction products formed by the thiosulfate-forming system
Amol System
S3062-
S20321.54 0.09 0.49
S2-
0.21 2.89 Completea 0.19 1.08 Minus FIH 0.38 0.17 Minus bisulfite reductase 0.18 0.22 1.72 Minus FII + boiled FlIb a Complete system consisted of the standard assay mixture plus FII. Bisulfite reductase concentration, 0.42 mg; FII, 1.4 mg. bFII was placed in a boiling-water bath for 5 min. TABLE 4. Degradation of [35S]thiosulfate formed by the thiosulfate-forming system' Counts/ Thiosulfate (counts/min) min de- % Recovery Expt graded S SO, 101 25,129 38,025 62,500 1 81 29,392 31,459 74,600 2 94 5,220 7,155 13,200 3 56 44 Avg distri-
bution a Reaction mixture was the same as that described for the complete system in Table 3. Bisulfite reductase concentration, 0.42 mg; FII, 1.4 mg; incubation time, 90 min.
DISCUSSION The results obtained in this study indicate that the bisulfite reductase, isolated from D.
vulgaris, reduces bisulfite to trithionate as the
major product and to sulfide as a minor product. Thiosulfate is not considered to be a product of the enzymatic process according to the observed labeling pattern. It is possible that the sulfane atom of this molecule originated from the proteins present in the reaction mixture. This explanation was given for the identical labeling pattern found in thiosulfate isolated from reaction mixtures containing C. pyrenoi-
dosa (13) and bisulfite reductase from D. nigriformed by D. vulgaris bisulfite reductase, it appears that this enzyme differs from its counterpart in D. nigrificans, which, reportedly,
ficans (2). Because small amounts of 3S2- are
does not reduce HMS03S2-- to 35S2--. Since it was previously demonstrated (6, 8) that the bisulfite reductase (desulfoviridin) from D. gigas and D. vulgaris formed trithionate and sulfide in varying amounts depending upon the assay condi-
tions, it is possible that the D. nigrificans bisulfite reductase (P582) can also reduce bisulfite
VOL. 126, 1976
BISULFITE REDUCTASE OF D. VULGARIS
to sulfide. Perhaps the conditions used in the studies by Akagi et al. (2) were unfavorable for sulfide formation. It can also be speculated that the desulfoviridin molecule contains an active site that can slowly reduce the intermediate species, between bisulfite and trithionate, to sulfide and that this property is not inherent to P582. Whatever the reason may be for this difference between the two bisulfite reductases, we believe that the main role for these enzymes is to form trithionate from bisulfite. Chambers and Trudinger (3) questioned the possibility that trithionate and thiosulfate are intermediates in dissimilatory sulfate reduction. Their conclusions were mainly based on studies involving washed and growing cells of D. desulfuricans and D. gigas. Whereas whole cell experiments can yield valuable information, there can occasionally be equivocal data obtained from such studies. Whole cells of D. desulfuricans and D. gigas were observed to reduce both sulfur atoms of thiosulfate at relatively equal rates (3). In addition, when 35SO42and unlabeled thiosulfate were incubated with D. desulfuricans cells the specific activity of the accumulated 35S2- was found not to be identical to that of the sulfane atom of thiosulfate. These data led Chambers and Trudinger (3) to conclude that thiosulfate was not an intermediate in the sulfate-reducing prpcess. Nakatsukasa and Akagi (15) observed that a partially purified thiosulfate reductase from D. nigrificans reduced both sulfur atoms of thiosulfate to sulfide at equal rates; however, a further purification of this enzyme showed that it reduced only the sulfane atom ofthiosulfate. It is, therefore, not surprising that whole cells can reduce the sulfane and sulfonate sulfur atoms to sulfide at equal rates. Furthermore, if two different substrates, such as sulfate and thiosulfate, are simultaneously incubated with whole cells to compare their rates of metabolism, it should be established that their rates of uptake are equal. On the basis of their conclusion that thiosulfate is not an intermediate in the sulfate reduction process, and since they observed that trithionate is reduced to thiosulfate, Chambers and Trudinger (3) suggested that trithionate is probably not an intermediate between bisulfite and sulfide. Because trithionate is a product of bisulfite reductase activity (5, 8-10, 12) and because we have evidence for the presence of a trithionate reductase activity in sulfate-reducing bacteria (unpublished data), we feel that one pathway for bisulfite reduction to sulfide involves trithionate and thiosulfate, as proposed by Kabayashi et al. (9). A second path-
737
way for sulfite reduction is catalyzed by an assimilatory sulfite reductase present in these organisms (11). Prior to this study we assumed that thiosulfate formation by bisulfite reductase plus FII (thiosulfate-forming system) occurred by the sequential reduction of bisulfite to trithionate to thiosulfate. However, the observations that this mixture did not reduce trithionate and the 35S-labeling pattern shown in Table 4 indicated that thiosulfate was formed through a pathway not involving trithionate. We propose that another pathway for bisulfite reduction, which involves the thiosulfate-forming system (18), is possible in D. vulgaris. We are currently attempting to purify the active protein of this system from the FII fraction. ACKNOWLEDGMENTS This study was supported in part by a grant, GB-37545, from the National Science Foundation, by Public Health Service grant AI-04672 from the National Institute of Allergy and Infectious Diseases, by Public Health Service training grant GM-703 from the National Institute of General Medical Sciences, and by grants from the University of Kansas Biomedical Sciences Support Committee and the General Research Fund. J. M. A. is a recipient of Research Career Development award GM-30,262 from the National Institute of General Medical Sciences.
LITERATURE CITED 1. Akagi, J. M., and L. L. Campbell. 1962. Studies on thermophilic sulfate-reducing bacteria. III. Adenosine triphosphate-sulfurylase of Clostridium nigrificans and Desulfovibrio desulfuricans. J. Bacteriol. 84:1194-1201. 2. Akagi, J. M., M. Chan, and V. Adams. 1974. Observations on the bisulfite reductase (P582) isolated from Desulfotomaculum nigrificans. J. Bacteriol. 120:240244. 3. Chambers, L. A., and P. A. Trudinger. 1975. Are thiosulfate and trithionate intermediates in dissimilatory sulfate reduction? J. Bacteriol. 123:36-40. 4. Fogo, J. K., and M. Popowski. 1949. Spectrophotometric determination of hydrogen sulfide. Anal. Chem. 21:732-734. 5. Jones, H. E., and G. W. Skyring. 1974. Reduction of sulfite to sulfide catalyzed by desulfoviridin from De-
sulfovibrio gigas. Aust. J. Biol. Sci. 27:7-14. 6. Jones, H. E., and G. W. Skyring. 1975. Effect of enzymic assay conditions on sulfite reduction catalyzed by desulfoviridin from Desulfovibrio gigas. Biochim. Biophys. Acta 377:52-60. 7. Kelly, D. P., L. A. Chambers, and P. A. Trudinger. 1969. Cyanolysis and spectrophotometric estimation of trithionate in mixture with thiosulfate and tetrathionate. Anal. Chem. 41:898-901. 8. Kobayashi, K., Y. Seki, and M. Ishimoto. 1974. Biochemical studies on sulfate reducing bacteria. XIII. Sulfite reductase from Desulfovibrio vulgaris. Mechanism of trithionate, thiosulfate, and sulfide formation and enzymatic properties. J. Biochem. (Tokyo) 75:519-529. 9. Kobayashi, K., S. Tachibana, and M. Ishimoto. 1969. Intermediary formation of trithionate in sulfite reduction by a sulfate-reducing bacterium. J. Biochem. (Tokyo) 65:155-157.
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DRAKE AND AKAGI
10. Kobayashi, K., E. Takahashi, and M. Ishimoto. 1972. Biochemical studies on sulfate-reducing bacteria. XI. Purification and some properties of sulfite reductase, desulfoviridin. J. Biochem. (Tokyo) 72:879-887. 11. Lee, J. P., J. LeGalland, and H. D. Peck. 1973. Isolation of assimilatory and dissimilatory-type sulfite reductases from Desulfovibrio vulgaris. J. Bacteriol. 115:529-542. 12. Lee, J. P., and H. D. Peck. 1971. Purification of the enzyme reducing bisulfite to trithionate from Desulfovibrio gigas and its identification as desulfoviridin.
Biochem. Biophys. Res. Commun. 45:583-589. 13. Levinthal, M., and J. A. Schiff. 1968. Studies on sulfate utilization by algae. 5. Identification of thiosulfate as the major acid-volatile product formed by a cell-free sulfate-reducing system from Chlorella. Plant Phys-
iol. 43:555-562. 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Nakatsukasa, W., and J. M. Akagi. 1969. Thiosulfate reductase isolated from Desulfotomaculum nigrificans. J. Bacteriol. 98:429-433. Postgate, J. R. 1959. A diagnostic reaction of Desulphovibrio desulphuricans. Nature (London) 183:481482. Samuelson, 0. 1963. Ion exchange separation in analytical chemistry, p. 416-418. John Wiley and Sons, Inc., New York. Suh, B., and J. M. Akagi. 1969. Formation of thiosulfate from sulfite by Desulfovibrio vulgaris. J. Bacteriol. 99:210-215.
14. Lowry,
15.
16. 17.
18.
