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[36] Enzymes involved in microbiological oxidation of thiosulfate and polythionates
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replicate samples with a slight excess of ethanolic iodine, which converts thiosulfate to tetrathionate. 355 in sulfate is then determined directly and the amount of [35S]thiosulfate present is given by the increase in the amount of [35S]tetrathionate found in the presence of iodine.
[36] E n z y m e s I n v o l v e d in Microbiological O x i d a t i o n of Thiosulfate and Polythionates
By DON P. KELLY and ANN P. WOOD Introduction Thiosulfate and polythionates ( S n O 6 2 - ) s e r v e as energy-yielding or electron-donating substrates in the metabolism of a wide range of chemolithotrophic and photolithotrophic bacteria, as well as some heterotrophs. 1-3 Relatively few enzymes have been positively implicated in these oxidative processes and only some of these have been highly purified and characterized. In this chapter we describe effective assay procedures for a variety of enzymes occurring in lithotrophs and some heterotrophs. Some of these enzymes [adenylylsulfate (APS) reductase and thiosulfate reductase] also occur in sulfate-reducing bacteria.
Methods
Thiosulfate Dehydrogenase [Thiosulfate : Cytochrome-c Oxidoreductase (Tetrathionate Synthesizing), Tetrathionate Synthase, ThiosulfateOxidizing Enzyme]: EC 1.8.2.2 Thiosulfate dehydrogenase occurs in several thiobacilli and some heterotrophs and catalyzes the oxidation 252032- -"> 54062- q- 2e252032- + (oxidized acceptor)
---> $ 4 0 6 2 -
-~-
(reduced acceptor)
i D. P. Kelly, in "Autotrophic Bacteria" (H. G. Schlegel and B. Bowien, eds.), p. 193. Science Tech Publ., Madison, Wisconsin, 1989. 2 D. P. Kelly, in "The Nitrogen and Sulphur Cycles" (J. A. Cole and S. J. Ferguson, eds.), p. 65. Cambridge Univ. Press, Cambridge, 1988. 3 D. P. Kelly, in "Bacterial Energetics" (T. A. Krulwich, ed.), p. 479. Academic Press, San Diego, 1990.
METHODS IN ENZYMOLOGY, VOL. 243
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The physiological acceptor is generally a cytochrome c, but the enzyme is commonly assayed with ferricyanide as the artificial acceptor: 2S2032- + 2Fe(CN)63- ~ 54062- Jr- 2Fe(CN)64Although intact cells of some thiobacilli, phototrophs and heterotrophs, as well as cell-free extracts and the purified enzyme, produce tetrathionate, there is some doubt that tetrathionate is necessarily always a normal free intermediate in thiosulfate or sulfide oxidation by normal cells. Verification of tetrathionate as the sole product of the reaction can be achieved either with [35]thiosulfate as substrate 4 followed by paper or thinlayer chromatography (see [35] in this volume) or by chemical analysis 5 (see [35] in this volume).
Reagents Potassium hydrogen phthalate-NaOH buffers (0.3 M), pH 4.5-6.0 Potassium phosphate buffers (0.3 M), pH 6.0-7.0 Potassium ferricyanide, 0.03 M Sodium thiosulfate, 0.1 M Procedure. The enzyme assay is based on that of Trudinger4'6 in which ferricyanide reduction is measured spectrophotometrically at 420 nm in a total volume of 3 ml in a 1-cm cuvette containing phthalate or phosphate buffer, pH 4.5-7.0 (300/zmol), N a 2 S 2 0 3 (30/zmol), K3Fe(CN)6 (3/zmol). The reaction is started by addition of cell-free extract or enzyme preparation. Decrease in absorbance at 420 nm is recorded and ferricyanide reduction estimated from a calibration curve or by using the millimolar extinction coefficient of 1.0. Enzyme activity is expressed as nanomoles of ferricyanide reduced per minute per milligram protein. In Thiobacillus tepidarius the specific activity measured between pH 4.5 and 7.0 showed the rate to be highest at the lowest p H . 7 Below pH 4.5 there is a chemical reaction between thiosulfate and fendcyanide that interferes with the assay. A similar assay procedure using one-third concentrations of reagents has been used with extracts of Thiobacillus neapolitanus, at pH values of 4.5-8.5, and again greatest activity was observed at the lowest pH. 8 The enzyme has also been successfully assayed using
4 p. A. Trudinger, Biochem. J. 78, 680 (1961). 5 W. P. Lu and D. P. Kelly, J. Gen. Microbiol. 134, 877 (1988). 6 p. A. Trudinger, Biochem. J. 78, 673 (1961). 7 A. P. Wood and D. P. Kelly, Arch. Microbiol. 144, 71 (1986). 8 j. Mason, D. P. Kelly, and A. P. Wood, J. Gen. Microbiol. 133, 1249 (1987).
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503
acetate buffer. 9 More recently the assay has been carried out in unbuffered ammonium sulfate solution with pH adjusted by addition of sulfuric acid.l° It may also be assayed using horse heart cytochrome c as electron acceptor. Here the reaction mixture (1 ml) contains 0.1 M potassium phosphate (pH 7.0), 1 /.tmol of Na2S203; 0.15 mg of cytochrome c, and 0.5-5.0 mg of protein. Cytochrome reduction is monitored as increase in absorbance at 550 nm. Purification ofThiosulfate Dehydrogenase. This enzyme has been purified from a number of different Thiobacillus spp. and found to vary greatly in structural and catalytic properties (for summary see Refs. 5 and 10). Thiosulfate dehydrogenase preparations from different thiobacilli have been shown to have molecular weight values of 102,000-138,000, and to be made up either of identical subunits (M r 45,000, T. tepidarius 5) or unequal subunits (Mr 24,000 and 20,000, Thiobacillus acidophilusl°). Most preparations were reported to contain no detectable heme 5but that purified 1026-fold from T. acidophilus contained about 5.3 mol of c-type heme per mole of native enzyme, made up of two different c-553 hemes, and is present in both subunit types) ° Thiosulfate dehydrogenase from T. acidophilus was purified to homogeneity in the following way. 1° All procedures are carried out at pH 7.0 and room temperature, except for the ammonium sulfate precipitation, which is done on ice. Cells are disrupted by a French pressure cell, extracted three times with culture supernatant, centrifuged (48,000 g, 20 min), and pooled. Ammonium sulfate to 3.0 M is added to the supernatant, precipitated protein centrifuged, redissolved in sodium citrate (25 mM, pH 7.0), recentrifuged, then subjected to hydrophobic interaction chromatography on a phenyl-Sepharose column combined with a fast protein liquid chromatography (FPLC) system. Ammonium sulfate is added (to 1.5 M) to the enzyme preparation, centrifuged (48,000 g, 30 min), then enzyme solution is loaded onto the column, equilibrated with 25 mM sodium citrate plus 1.5 M ammonium sulfate, pH 7.0, and the column eluted with equilibration buffer at 3 ml/min until the absorbance of the eluant at 280 nm is less than 0.2. A linear gradient of 1.5 to 0 M ammonium sulfate in citrate is then applied. Fractions containing enzyme activity are pooled, concentrated, and desalted to a final concentration of 25 mM ammonium sulfate (Centriprep-30; Amicon, Danvers, MA). This is further purified by anion-exchange FPLC. After loading on a Mono Q column 9 A. J. Smith, J. Gen. Microbiol. 42, 371 (1966). I0 R. Meulenberg, J. T. Pronk, W. Hazeu, J. P. van Dijken, J. Frank, P. Bos, and J. G. Kuenen, J. Gen. Microbiol. 139, 2033-2039 (1993).
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equilibrated with sodium citrate (25 mM, pH 7.0), and washing with buffer until the eluant shows an absorbance at 280 nm of less than 0.0005, a linear gradient of 0 to 1.0 M NaC1 in citrate buffer is applied. Enzymecontaining fractions are further purified by gel filtration on a Superose6 column equilibrated with 0.5 M ammonium sulfate. Protein elution is monitored at 280 nm, and active fractions are pooled, concentrated, and stored in liquid nitrogen. This produces about 1000-fold purification, giving a product that is homogeneous in gel filtration.I° The enzyme in T. tepidarius was located in the periplasm of the cells and could be released by lysozyme treatment and osmotic shock).
Trithionate Hydrolase (Trithionate Thiosulfohydrolase): EC 3.12.1.1 - O 3 S - S - S O 3- + H 2 0 ~
- S - S O 3- + H2SO 4
The activity of trithionate hydrolase was first demonstrated in T. neapolitanus and the enzyme subsequently was recovered from both T. tepidarius and T. acidophilus. TM Procedure 1. The enzyme can be determined in a coupled assay with thiosulfate dehydrogenase (see above), using 0.1 mM sodium trithionate instead of thiosulfate, and measuring thiosulfate-dependent cytochrome c reduction at pH 7.0. 5 Thiosulfate produced by the hydrolase becomes the substrate for an excess of thiosulfate dehydrogenase and the rate of reduction of cytochrome c equates to the specific activity of the hydrolase. Trithionate solutions are prepared in 0.1 M potassium phosphate, pH 7.0, and used within 5 hr to minimize chemical hydrolysis. Procedure 2. Trithionate hydrolase can also be measured in a discontinuous assay 11 in a well-mixed, thermostatted reaction chamber (10 ml) containing cell-free extract in 25 mMpotassium phosphate and 1 M ammonium sulfate, pH 3.0. The reaction is started by adding 1 mM trithionate and 0.5-ml samples removed at intervals into 0.1 ml of 0.125 M potassium cyanide for thiosulfate determination by a modification of the cyanolysis method (see [35] in this volume). Following addition of 0.1 ml of 0.075 M copper chloride, 1.0 ml of 0.3 M ferric nitrate in 3 M HNO 3 is added and ferric thiocyanate color measured at 460 nm. Ferric nitrate is used in this assay at a concentration 10-fold higher than in the standard method (see [35] in this volume) to overcome inhibition of color development by the high ammonium sulfate concentrations present in the samples. u R. Meulenberg, J. T. Pronk, J. Frank, W. Hazeu, P. Bos, and J. G. Kuenen, Fur. J. Biochem. 2119, 367 (1992).
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Rhodanese (Thiosulfate:Cyanide Sulfurtransferase): EC 2.8.1.1 Rhodanese catalyzes transfer of the sulfane sulfur of thiosulfate to an acceptor, which is normally cyanide in the standard assay, and is likely to be cyanide under some physiological conditions. -S-SO3- + CN- ~ SCN-
+ 8032-
Rhodanese has been found in representatives of all the kingdoms. It can transfer the sulfane sulfur to other acceptors, including lipoic acid. 12
Procedure I: Discontinuous Determination of Thiocyanate Product Reagents Tris-HCl buffers (1.0 M), pH 7.6-10.6 Sodium thiosulfate, 1.0 M Potassium cyanide, 1.0 M Formaldehyde solution (formalin), 38% (w/v) Ferric reagent: 16% (w/v) ferric nitrate (nonahydrate) in 1.0 M nitric acid Reaction mixtures in replicate small test tubes contain 0.25 ml of Tris buffer, 0.05 ml of thiosulfate, 2.15 ml of water, and cell-free extract. 13,14 After incubation for 2-3 min, 0.05 ml of KCN is added and mixed rapidly. The reaction is stopped at timed intervals by addition of 0.2 ml of 38% (w/v) formaldehyde to each tube, followed by immediate mixing. Addition of 1.3 ml of 16% (w/v) ferric nitrate in 1 M nitric acid to each tube produces ferric thiocyanate, which is measured by absorbance at 470 nm and its concentration calculated by reference to a standard curve. Activity is expressed as nanomoles of thiocyanate formed per minute per milligram protein.
Procedure 2: Continuous Spectrophotometric Determination of Sulfite Production by Coupled Oxidation of 2,6-Dichlorophenol-indophenol Reagents Tris-HCl buffer (0.2 M) pH 8.6 Sodium thiosulfate, 0.3 M 2,6-Dichlorophenolindophenol (DCPIP), 0.004 M 12 M. Silver and D. P. Kelly, J. Gen. Microbiol. 97, 277 (1976). 13 B. S6rbo, Acta Chem. Scand. 7, 1129 (1953). 14 T. J. Bowen, P. J. Butler, and F. C. Happold, Biochem. J. 97, 651 (1965).
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METABOLISM OF POLYTHIONATES
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N-Methylphenazonium methosulfate (PMS), 5 mg/ml KCN, 2.0 M Typically a final reaction mixture volume of 3 ml in a 1-cm cuvette will contain 1.5 ml of Tris buffer (300/xmol), 0.5 ml of thiosulfate (150 gmol), 0.05 ml of DCPIP (0.2/zmol), 0. I ml of PMS, cell-free extract, and water to a total of 2.95 ml. ~5-17A blank treatment is prepared without DCPIP and extract. Assays can be run at the temperature within the spectrophotometer cell holder or at 30° in a controlled temperature holder. After incubation for 2-3 min, reaction is initiated by rapid addition and mixing of 0.05 ml of KCN (100/zmol). Decrease in absorbance at 600 nm measures DCPIP reduction and enzyme activity is expressed as nanomoles of DCPIP reduced per minute per milliliter protein. The 600-nm millimolar extinction coefficient for DCPIP is 20.6.18 It should be noted that the addition of cyanide raises the actual pH of the mixtures to about pH 9.3. For determination of pH optima or assay ofrhodanese enzymes with pH optima higher than pH 9.3, different buffers (e.g., glycine-NaOH) including Tris (without acid, pH 10.6) can be used. 19
Thiocyanate Hydrolase SCN- + 2H20 ,~ COS + NH 3 + OHThiocyanate hydrolase has to date been demonstrated only in Thiobacillus thioparus, in which it is induced by growth on thiocyanate, and was purified 52-fold by ammonium sulfate precipitation, and DEAE-Sephacel and hydroxylapatite column chromatography. 2° It is made up of three different subunits ( M r 19,000, 23,000, and 32,000) and the native enzyme has a molecular weight of 126,000. Activity is optimal at pH 7.5-8.0, 30-40 °, and has a K m of about 11 mM. Assay Procedure. Activity can be determined by incubating a cell-free extract in 0.1 M phosphate buffer, pH 7.5, with 20 mM KSCN at 30° and measuring the disappearance of either or both thiocyanates (see [35] in this volume) and ammonia formation (by standard Nessler procedure) either after 20 min 2° or at suitable time intervals to obtain a reaction time course. The production of COS can be demonstrated by conducting the 15 A. J. Smith and J. Lascelles, J. Gen. Microbiol. 42, 357 (1966). 16 D. P. Kelly, Arch. Mikrobiol. 61, 59 (1968). 17 W. P. Lu and D. P. Kelly, FEMS Microbiol. Lett. 18, 289 (1983). 18 M. A. Steinmetz and U. Fischer, Arch. Microbiol. 142, 253 (1985). 19 A. P. Wood and D. P. Kelly, J. Gen. Microbiol. 125, 55 (1981). 2o y . Katayama, Y. Narahara, Y. Inoue, F. Amano, T. Kanagawa,and H. Kuraishi, J. Biol. Chem. 267, 9170 (1992).
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507
assay in a sealed vessel and sampling the head space for analysis by gas chromatography.
Thiosulfate Reductase (Thiosulfate-Thiol Transferase or Sulfurtransferase): Glutathione Dependent (EC 2.8.1.3) and Dithiothreitol Dependent (EC 2.8.1.5) - S - - S O 3- +
2e- ~
S 2- + S O s 2-
Thiosulfate reductase can be assayed using reduced methyl viologen as the reductant, with continuous spectrophotometric measurement of dye oxidation, m
Reagents Tris-acetate buffer (pH 8.7), 1.0 M Reduced methyl viologen, 2 mM in 0.05 M Tris-acetate, pH 8.7 Sodium thiosulfate, 0.05 M Procedure. Reaction is carried out in a cuvette that can be stoppered with a serum cap: 0.05 ml of Tris buffer and 0.05-0.2 ml of cell-free extract are mixed and made up to 0.4 ml with water. The cuvette is stoppered, deaerated under vacuum for 10 min, and regassed with oxygen-free nitrogen, then 0.5 ml of reduced methyl viologen is added by Hamilton syringe. Reaction is started by injecting 0. I ml of thiosulfate and the oxidation of reduced methyl viologen followed at 600 nm, using a millimolar extinction coefficient of 113. TM AMP-Dependent and AMP-Independent Oxidation of Sulfite A number of enzymes whose activities result in the conversion of sulfite to sulfate are known. We describe assay of (1) "sulfite dehydrogenase" and (2) "APS reductase." SO32- + H20 ~ 8042- + 2H + + 2e-
.
SO3 2- + AMP ~ - A P S + 2e-
2.
Adenylylsulfate can be converted to sulfate by the action of ADP- or ATP-sulfurylase: APS + PO43- (or P2074-)
~
SO4 2- +
ADP (or ATP)
Reagents Tris-HCl or Tris-H2SO 4 buffers (0.3 M), pH 7.4, 8.0, or 8.4 Potassium ferficyanide, 0.03 M
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METABOLISM OF POLYTHIONATES
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Sodium sulfite, 0.18 M, freshly prepared in 0.1 M Tris-HCl, pH 7.8, containing 10 mM ethylenediaminetetraacetic acid (EDTA) AMP, 0.045 M
Sulfite Dehydrogenase (Sulfite:Ferricytochrome-c Oxidoreductase, Sulfite Oxidase): EC 1.8.2.1 SO32- + 2Fe(CN)63- + H20 ~ SO42- + 2Fe(CN)64- + 2H ÷ Sulfite-dependent reduction of ferricyanide can be measured in reaction mixtures (3 ml) containing 1 ml of Tris buffer (300/zmol), 0.1 ml of potassium ferricyanide (3/zmol), 0.05 ml of Na2SO 3 (9/xmol), and protein (1-3 mg). The reaction is started by adding enzyme or sulfite, and decrease in absorbance at 420 nm recorded, using buffer plus water as a blank. The millimolar extinction coefficient is 1.0. The rates of sulfite autooxidation (before enzyme addition) and any endogenous reduction of ferricyanide by the enzyme preparation (in the absence of sulfite) need to be assayed as controls. The enzyme can also be assayed using horse heart cytochrome c as electron acceptor, using the same procedure as for thiosulfate dehydrogenase (above) but with sulfite as substrate, zl Preparations of this enzyme purified 2000-fold from Thiobacillus versutus showed it to be a monomer (Mr 44,000) and to contain intimately associated cytochrome c-551.21
Adenylylsulfate Reductase: EC 1.8.99.2 Adenylylsulfate reductase activity is estimated from the difference in rate of reduction of ferricyanide (A4z0) in the assay described above, in the presence of sulfite with and without AMP. z2 Assays (3 ml in 1-cm cuvettes) contain 0.1 M Tris-HCl (pH 7.4), 1 mM potassium ferricyanide, 3 mM sodium sulfite, and cell-free extract. The rate of ferricyanide reduction due to AMP-independent sulfite oxidation is determined for a few minutes before 0.05 ml of AMP (0.75 mM) is added to the cuvette and the rate of ferricyanide reduction measured for a further 5-10 min. Any stimulation of the rate of ferricyanide reduction by AMP is a measure of sulfite oxidation that can be ascribed to APS reductase. Controls should omit sulfite or AMP. The sequence of additions of AMP, sulfite, and extract should be tested to ensure that results obtained are independent of the order of exposure of the enzymes to the substrates. 21 W.-P. Lu and D. P. Kelly, J. Gen. Microbiol. 130, 1683 (1984). 22 T. J. Bowen, F. C.Happold, and B. F. Taylor, Biochim. Biophys. Acta 118, 566 (1966).
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Sulfur Oxygenase (Sulfur Dioxygenase, Sulfur:Oxygen Oxidoreductase, Sulfur-Oxidizing Enzyme): EC 1.13.11.18 S 8 + 8H20 + 802 ~- 8H2503
Sulfur oxygenase is assayed by determination of oxygen uptake and thiosulfate production in the presence of powdered sulfur and reduced glutathione. 23,24 Respirometric assays are conducted in an oxygen electrode cell using reaction mixtures (2 ml) containing 0.25 M Tris-HC1 (pH 7.8), sulfur (48 mg), catalase (0.25 mg), 2,2'-bipyridyl (0.1 mM), reduced glutathione (2.5 mM), and crude cell-free extract (0.05-0.25 ml). Oxygen uptake rates should be corrected for (low) chemical control oxidation rates. Activities measured at 30° with cell-free extracts of thiobacilli are generally low, requiring incubation periods of an hour or longer. The time course of thiosulfate formation in an identical assay mixture can be followed in small flasks shaken in air and sampled at intervals up to 3 hr. The samples (0.1 ml) are mixed with 0.1 ml of 1 M cadmium acetate and then assayed cyanolytically for thiosulfate (see [35] in this volume).
Glutathione-Independent Sulfur "Oxidase" Sulfur oxidation in cell-free extracts of some thiobacilli is seen in mixtures lacking glutathione, and is apparently due to an uncharacterized enzyme(s) that might link oxidation to electron transport, rather than to being an oxygenase25: S 8 + 24H20 ~- 8H2SO 3 + 32H + + 32eActivity can be measured in an oxygen electrode cell using an assay mixture (2 ml) containing 0.1 M Tris-HC1 (pH 8.0), powdered sulfur (30 mg), with and without Tween 80 (1%, w/v). Taylor z5 used about 25 mg of protein per assay and found that at least 15 mg was required to initiate the reaction.
Proteins A and B and Thiosulfate-Oxidizing Multienzyme System of Thiobacillus versutus An enzyme system located in the periplasmic space of T. uersutus is capable of the complete oxidation of thiosulfate to sulfate, without the intermediate formation or accumulation of polythionates, with the coupled reduction of several cytochromes. 26 This can be assayed only with a 23 I. Suzuki, Biochim. Biophys. Acta 1114, 359 (1965). 24 I. Suzuki and M. Silver, Biochim. Biophys. Acta 122, 22 (1966). 25 B° F. Taylor, Biochim. Biophys. Acta 170, 112 (1968). 26 W.-P. Lu, B. E. P. Swoboda, and D. P. Kelly, Biochim. Biophys. Acta 828, 116 (1985).
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complete system comprising at least two proteins (Mr 16,000 and 64,000) and two unusual c-type cytochromes. One protein (A) is a thiosulfatebinding enzyme and the other (B) contains an unusual binuclear manganese cluster. The mechanisms of action of these enzymes is unknown and the activity of the components of the system cannot be assayed independently. Their assay and properties are therefore not further described in this chapter, but the specialist reader is referred to the original literature. 26-31 27 W.-P. Lu and D. P. Kelly, J. Gen. Microbiol. 129, 1673 (1983). 28 W.-P. Lu and D. P. Kelly, J. Gen. Microbiol. 129, 3549 (1983). 29 W.-P. Lu and D. P. Kelly, Biochim. Biophys. Acta 765, 106 (1984). 3o W.-P. Lu, FEMS Microbiol. Lett. 34, 313 (1986). 31 R. Cammack, A. Chapman, W.-P. Lu, A. Karagouni, and D. P. Kelly, FEBS Lett. 253, 239 (1989).
[37] W h o l e - O r g a n i s m M e t h o d s for I n o r g a n i c Sulfur Oxidation b y C h e m o l i t h o t r o p h s a n d P h o t o l i t h o t r o p h s By DON P. KELLY and ANN P. WOOD Introduction This chapter describes some of the techniques that have been used in studies of sulfur compound oxidation and energy coupling using suspensions of intact organisms and for the separate identification and characterization of enzymes that are located in the periplasm, rather than the cytoplasm, of thiobacilli. In some cases the techniques are derived from those developed for studies on other microorganisms or using mitochondria, and are outlined primarily to demonstrate that the same principles apply to the less routinely studied lithotrophs. Some of the procedures provide examples of the use of the analytical methods described in [35] in this volume.
Demonstration of Periplasmic Location of Some Enzymes Involved in Sulfur Compound Oxidation in Thiobacilli Separation of the periplasmic fractions from both Thiobacillus versutus and Thiobacillus tepidarius has been achieved by methods based on a METHODS IN ENZYMOLOGY, VOL. 243
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
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replicate samples with a slight excess of ethanolic iodine, which converts thiosulfate to tetrathionate. 355 in sulfate is then determined directly and the amount of [35S]thiosulfate present is given by the increase in the amount of [35S]tetrathionate found in the presence of iodine.
[36] E n z y m e s I n v o l v e d in Microbiological O x i d a t i o n of Thiosulfate and Polythionates
By DON P. KELLY and ANN P. WOOD Introduction Thiosulfate and polythionates ( S n O 6 2 - ) s e r v e as energy-yielding or electron-donating substrates in the metabolism of a wide range of chemolithotrophic and photolithotrophic bacteria, as well as some heterotrophs. 1-3 Relatively few enzymes have been positively implicated in these oxidative processes and only some of these have been highly purified and characterized. In this chapter we describe effective assay procedures for a variety of enzymes occurring in lithotrophs and some heterotrophs. Some of these enzymes [adenylylsulfate (APS) reductase and thiosulfate reductase] also occur in sulfate-reducing bacteria.
Methods
Thiosulfate Dehydrogenase [Thiosulfate : Cytochrome-c Oxidoreductase (Tetrathionate Synthesizing), Tetrathionate Synthase, ThiosulfateOxidizing Enzyme]: EC 1.8.2.2 Thiosulfate dehydrogenase occurs in several thiobacilli and some heterotrophs and catalyzes the oxidation 252032- -"> 54062- q- 2e252032- + (oxidized acceptor)
---> $ 4 0 6 2 -
-~-
(reduced acceptor)
i D. P. Kelly, in "Autotrophic Bacteria" (H. G. Schlegel and B. Bowien, eds.), p. 193. Science Tech Publ., Madison, Wisconsin, 1989. 2 D. P. Kelly, in "The Nitrogen and Sulphur Cycles" (J. A. Cole and S. J. Ferguson, eds.), p. 65. Cambridge Univ. Press, Cambridge, 1988. 3 D. P. Kelly, in "Bacterial Energetics" (T. A. Krulwich, ed.), p. 479. Academic Press, San Diego, 1990.
METHODS IN ENZYMOLOGY, VOL. 243
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
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The physiological acceptor is generally a cytochrome c, but the enzyme is commonly assayed with ferricyanide as the artificial acceptor: 2S2032- + 2Fe(CN)63- ~ 54062- Jr- 2Fe(CN)64Although intact cells of some thiobacilli, phototrophs and heterotrophs, as well as cell-free extracts and the purified enzyme, produce tetrathionate, there is some doubt that tetrathionate is necessarily always a normal free intermediate in thiosulfate or sulfide oxidation by normal cells. Verification of tetrathionate as the sole product of the reaction can be achieved either with [35]thiosulfate as substrate 4 followed by paper or thinlayer chromatography (see [35] in this volume) or by chemical analysis 5 (see [35] in this volume).
Reagents Potassium hydrogen phthalate-NaOH buffers (0.3 M), pH 4.5-6.0 Potassium phosphate buffers (0.3 M), pH 6.0-7.0 Potassium ferricyanide, 0.03 M Sodium thiosulfate, 0.1 M Procedure. The enzyme assay is based on that of Trudinger4'6 in which ferricyanide reduction is measured spectrophotometrically at 420 nm in a total volume of 3 ml in a 1-cm cuvette containing phthalate or phosphate buffer, pH 4.5-7.0 (300/zmol), N a 2 S 2 0 3 (30/zmol), K3Fe(CN)6 (3/zmol). The reaction is started by addition of cell-free extract or enzyme preparation. Decrease in absorbance at 420 nm is recorded and ferricyanide reduction estimated from a calibration curve or by using the millimolar extinction coefficient of 1.0. Enzyme activity is expressed as nanomoles of ferricyanide reduced per minute per milligram protein. In Thiobacillus tepidarius the specific activity measured between pH 4.5 and 7.0 showed the rate to be highest at the lowest p H . 7 Below pH 4.5 there is a chemical reaction between thiosulfate and fendcyanide that interferes with the assay. A similar assay procedure using one-third concentrations of reagents has been used with extracts of Thiobacillus neapolitanus, at pH values of 4.5-8.5, and again greatest activity was observed at the lowest pH. 8 The enzyme has also been successfully assayed using
4 p. A. Trudinger, Biochem. J. 78, 680 (1961). 5 W. P. Lu and D. P. Kelly, J. Gen. Microbiol. 134, 877 (1988). 6 p. A. Trudinger, Biochem. J. 78, 673 (1961). 7 A. P. Wood and D. P. Kelly, Arch. Microbiol. 144, 71 (1986). 8 j. Mason, D. P. Kelly, and A. P. Wood, J. Gen. Microbiol. 133, 1249 (1987).
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acetate buffer. 9 More recently the assay has been carried out in unbuffered ammonium sulfate solution with pH adjusted by addition of sulfuric acid.l° It may also be assayed using horse heart cytochrome c as electron acceptor. Here the reaction mixture (1 ml) contains 0.1 M potassium phosphate (pH 7.0), 1 /.tmol of Na2S203; 0.15 mg of cytochrome c, and 0.5-5.0 mg of protein. Cytochrome reduction is monitored as increase in absorbance at 550 nm. Purification ofThiosulfate Dehydrogenase. This enzyme has been purified from a number of different Thiobacillus spp. and found to vary greatly in structural and catalytic properties (for summary see Refs. 5 and 10). Thiosulfate dehydrogenase preparations from different thiobacilli have been shown to have molecular weight values of 102,000-138,000, and to be made up either of identical subunits (M r 45,000, T. tepidarius 5) or unequal subunits (Mr 24,000 and 20,000, Thiobacillus acidophilusl°). Most preparations were reported to contain no detectable heme 5but that purified 1026-fold from T. acidophilus contained about 5.3 mol of c-type heme per mole of native enzyme, made up of two different c-553 hemes, and is present in both subunit types) ° Thiosulfate dehydrogenase from T. acidophilus was purified to homogeneity in the following way. 1° All procedures are carried out at pH 7.0 and room temperature, except for the ammonium sulfate precipitation, which is done on ice. Cells are disrupted by a French pressure cell, extracted three times with culture supernatant, centrifuged (48,000 g, 20 min), and pooled. Ammonium sulfate to 3.0 M is added to the supernatant, precipitated protein centrifuged, redissolved in sodium citrate (25 mM, pH 7.0), recentrifuged, then subjected to hydrophobic interaction chromatography on a phenyl-Sepharose column combined with a fast protein liquid chromatography (FPLC) system. Ammonium sulfate is added (to 1.5 M) to the enzyme preparation, centrifuged (48,000 g, 30 min), then enzyme solution is loaded onto the column, equilibrated with 25 mM sodium citrate plus 1.5 M ammonium sulfate, pH 7.0, and the column eluted with equilibration buffer at 3 ml/min until the absorbance of the eluant at 280 nm is less than 0.2. A linear gradient of 1.5 to 0 M ammonium sulfate in citrate is then applied. Fractions containing enzyme activity are pooled, concentrated, and desalted to a final concentration of 25 mM ammonium sulfate (Centriprep-30; Amicon, Danvers, MA). This is further purified by anion-exchange FPLC. After loading on a Mono Q column 9 A. J. Smith, J. Gen. Microbiol. 42, 371 (1966). I0 R. Meulenberg, J. T. Pronk, W. Hazeu, J. P. van Dijken, J. Frank, P. Bos, and J. G. Kuenen, J. Gen. Microbiol. 139, 2033-2039 (1993).
504
METABOLISM OF POLYTHIONATES
[36]
equilibrated with sodium citrate (25 mM, pH 7.0), and washing with buffer until the eluant shows an absorbance at 280 nm of less than 0.0005, a linear gradient of 0 to 1.0 M NaC1 in citrate buffer is applied. Enzymecontaining fractions are further purified by gel filtration on a Superose6 column equilibrated with 0.5 M ammonium sulfate. Protein elution is monitored at 280 nm, and active fractions are pooled, concentrated, and stored in liquid nitrogen. This produces about 1000-fold purification, giving a product that is homogeneous in gel filtration.I° The enzyme in T. tepidarius was located in the periplasm of the cells and could be released by lysozyme treatment and osmotic shock).
Trithionate Hydrolase (Trithionate Thiosulfohydrolase): EC 3.12.1.1 - O 3 S - S - S O 3- + H 2 0 ~
- S - S O 3- + H2SO 4
The activity of trithionate hydrolase was first demonstrated in T. neapolitanus and the enzyme subsequently was recovered from both T. tepidarius and T. acidophilus. TM Procedure 1. The enzyme can be determined in a coupled assay with thiosulfate dehydrogenase (see above), using 0.1 mM sodium trithionate instead of thiosulfate, and measuring thiosulfate-dependent cytochrome c reduction at pH 7.0. 5 Thiosulfate produced by the hydrolase becomes the substrate for an excess of thiosulfate dehydrogenase and the rate of reduction of cytochrome c equates to the specific activity of the hydrolase. Trithionate solutions are prepared in 0.1 M potassium phosphate, pH 7.0, and used within 5 hr to minimize chemical hydrolysis. Procedure 2. Trithionate hydrolase can also be measured in a discontinuous assay 11 in a well-mixed, thermostatted reaction chamber (10 ml) containing cell-free extract in 25 mMpotassium phosphate and 1 M ammonium sulfate, pH 3.0. The reaction is started by adding 1 mM trithionate and 0.5-ml samples removed at intervals into 0.1 ml of 0.125 M potassium cyanide for thiosulfate determination by a modification of the cyanolysis method (see [35] in this volume). Following addition of 0.1 ml of 0.075 M copper chloride, 1.0 ml of 0.3 M ferric nitrate in 3 M HNO 3 is added and ferric thiocyanate color measured at 460 nm. Ferric nitrate is used in this assay at a concentration 10-fold higher than in the standard method (see [35] in this volume) to overcome inhibition of color development by the high ammonium sulfate concentrations present in the samples. u R. Meulenberg, J. T. Pronk, J. Frank, W. Hazeu, P. Bos, and J. G. Kuenen, Fur. J. Biochem. 2119, 367 (1992).
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505
Rhodanese (Thiosulfate:Cyanide Sulfurtransferase): EC 2.8.1.1 Rhodanese catalyzes transfer of the sulfane sulfur of thiosulfate to an acceptor, which is normally cyanide in the standard assay, and is likely to be cyanide under some physiological conditions. -S-SO3- + CN- ~ SCN-
+ 8032-
Rhodanese has been found in representatives of all the kingdoms. It can transfer the sulfane sulfur to other acceptors, including lipoic acid. 12
Procedure I: Discontinuous Determination of Thiocyanate Product Reagents Tris-HCl buffers (1.0 M), pH 7.6-10.6 Sodium thiosulfate, 1.0 M Potassium cyanide, 1.0 M Formaldehyde solution (formalin), 38% (w/v) Ferric reagent: 16% (w/v) ferric nitrate (nonahydrate) in 1.0 M nitric acid Reaction mixtures in replicate small test tubes contain 0.25 ml of Tris buffer, 0.05 ml of thiosulfate, 2.15 ml of water, and cell-free extract. 13,14 After incubation for 2-3 min, 0.05 ml of KCN is added and mixed rapidly. The reaction is stopped at timed intervals by addition of 0.2 ml of 38% (w/v) formaldehyde to each tube, followed by immediate mixing. Addition of 1.3 ml of 16% (w/v) ferric nitrate in 1 M nitric acid to each tube produces ferric thiocyanate, which is measured by absorbance at 470 nm and its concentration calculated by reference to a standard curve. Activity is expressed as nanomoles of thiocyanate formed per minute per milligram protein.
Procedure 2: Continuous Spectrophotometric Determination of Sulfite Production by Coupled Oxidation of 2,6-Dichlorophenol-indophenol Reagents Tris-HCl buffer (0.2 M) pH 8.6 Sodium thiosulfate, 0.3 M 2,6-Dichlorophenolindophenol (DCPIP), 0.004 M 12 M. Silver and D. P. Kelly, J. Gen. Microbiol. 97, 277 (1976). 13 B. S6rbo, Acta Chem. Scand. 7, 1129 (1953). 14 T. J. Bowen, P. J. Butler, and F. C. Happold, Biochem. J. 97, 651 (1965).
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METABOLISM OF POLYTHIONATES
[36l
N-Methylphenazonium methosulfate (PMS), 5 mg/ml KCN, 2.0 M Typically a final reaction mixture volume of 3 ml in a 1-cm cuvette will contain 1.5 ml of Tris buffer (300/xmol), 0.5 ml of thiosulfate (150 gmol), 0.05 ml of DCPIP (0.2/zmol), 0. I ml of PMS, cell-free extract, and water to a total of 2.95 ml. ~5-17A blank treatment is prepared without DCPIP and extract. Assays can be run at the temperature within the spectrophotometer cell holder or at 30° in a controlled temperature holder. After incubation for 2-3 min, reaction is initiated by rapid addition and mixing of 0.05 ml of KCN (100/zmol). Decrease in absorbance at 600 nm measures DCPIP reduction and enzyme activity is expressed as nanomoles of DCPIP reduced per minute per milliliter protein. The 600-nm millimolar extinction coefficient for DCPIP is 20.6.18 It should be noted that the addition of cyanide raises the actual pH of the mixtures to about pH 9.3. For determination of pH optima or assay ofrhodanese enzymes with pH optima higher than pH 9.3, different buffers (e.g., glycine-NaOH) including Tris (without acid, pH 10.6) can be used. 19
Thiocyanate Hydrolase SCN- + 2H20 ,~ COS + NH 3 + OHThiocyanate hydrolase has to date been demonstrated only in Thiobacillus thioparus, in which it is induced by growth on thiocyanate, and was purified 52-fold by ammonium sulfate precipitation, and DEAE-Sephacel and hydroxylapatite column chromatography. 2° It is made up of three different subunits ( M r 19,000, 23,000, and 32,000) and the native enzyme has a molecular weight of 126,000. Activity is optimal at pH 7.5-8.0, 30-40 °, and has a K m of about 11 mM. Assay Procedure. Activity can be determined by incubating a cell-free extract in 0.1 M phosphate buffer, pH 7.5, with 20 mM KSCN at 30° and measuring the disappearance of either or both thiocyanates (see [35] in this volume) and ammonia formation (by standard Nessler procedure) either after 20 min 2° or at suitable time intervals to obtain a reaction time course. The production of COS can be demonstrated by conducting the 15 A. J. Smith and J. Lascelles, J. Gen. Microbiol. 42, 357 (1966). 16 D. P. Kelly, Arch. Mikrobiol. 61, 59 (1968). 17 W. P. Lu and D. P. Kelly, FEMS Microbiol. Lett. 18, 289 (1983). 18 M. A. Steinmetz and U. Fischer, Arch. Microbiol. 142, 253 (1985). 19 A. P. Wood and D. P. Kelly, J. Gen. Microbiol. 125, 55 (1981). 2o y . Katayama, Y. Narahara, Y. Inoue, F. Amano, T. Kanagawa,and H. Kuraishi, J. Biol. Chem. 267, 9170 (1992).
[36]
OXIDATION OF THIOSULFATE AND POLYTHIONATES
507
assay in a sealed vessel and sampling the head space for analysis by gas chromatography.
Thiosulfate Reductase (Thiosulfate-Thiol Transferase or Sulfurtransferase): Glutathione Dependent (EC 2.8.1.3) and Dithiothreitol Dependent (EC 2.8.1.5) - S - - S O 3- +
2e- ~
S 2- + S O s 2-
Thiosulfate reductase can be assayed using reduced methyl viologen as the reductant, with continuous spectrophotometric measurement of dye oxidation, m
Reagents Tris-acetate buffer (pH 8.7), 1.0 M Reduced methyl viologen, 2 mM in 0.05 M Tris-acetate, pH 8.7 Sodium thiosulfate, 0.05 M Procedure. Reaction is carried out in a cuvette that can be stoppered with a serum cap: 0.05 ml of Tris buffer and 0.05-0.2 ml of cell-free extract are mixed and made up to 0.4 ml with water. The cuvette is stoppered, deaerated under vacuum for 10 min, and regassed with oxygen-free nitrogen, then 0.5 ml of reduced methyl viologen is added by Hamilton syringe. Reaction is started by injecting 0. I ml of thiosulfate and the oxidation of reduced methyl viologen followed at 600 nm, using a millimolar extinction coefficient of 113. TM AMP-Dependent and AMP-Independent Oxidation of Sulfite A number of enzymes whose activities result in the conversion of sulfite to sulfate are known. We describe assay of (1) "sulfite dehydrogenase" and (2) "APS reductase." SO32- + H20 ~ 8042- + 2H + + 2e-
.
SO3 2- + AMP ~ - A P S + 2e-
2.
Adenylylsulfate can be converted to sulfate by the action of ADP- or ATP-sulfurylase: APS + PO43- (or P2074-)
~
SO4 2- +
ADP (or ATP)
Reagents Tris-HCl or Tris-H2SO 4 buffers (0.3 M), pH 7.4, 8.0, or 8.4 Potassium ferficyanide, 0.03 M
508
METABOLISM OF POLYTHIONATES
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Sodium sulfite, 0.18 M, freshly prepared in 0.1 M Tris-HCl, pH 7.8, containing 10 mM ethylenediaminetetraacetic acid (EDTA) AMP, 0.045 M
Sulfite Dehydrogenase (Sulfite:Ferricytochrome-c Oxidoreductase, Sulfite Oxidase): EC 1.8.2.1 SO32- + 2Fe(CN)63- + H20 ~ SO42- + 2Fe(CN)64- + 2H ÷ Sulfite-dependent reduction of ferricyanide can be measured in reaction mixtures (3 ml) containing 1 ml of Tris buffer (300/zmol), 0.1 ml of potassium ferricyanide (3/zmol), 0.05 ml of Na2SO 3 (9/xmol), and protein (1-3 mg). The reaction is started by adding enzyme or sulfite, and decrease in absorbance at 420 nm recorded, using buffer plus water as a blank. The millimolar extinction coefficient is 1.0. The rates of sulfite autooxidation (before enzyme addition) and any endogenous reduction of ferricyanide by the enzyme preparation (in the absence of sulfite) need to be assayed as controls. The enzyme can also be assayed using horse heart cytochrome c as electron acceptor, using the same procedure as for thiosulfate dehydrogenase (above) but with sulfite as substrate, zl Preparations of this enzyme purified 2000-fold from Thiobacillus versutus showed it to be a monomer (Mr 44,000) and to contain intimately associated cytochrome c-551.21
Adenylylsulfate Reductase: EC 1.8.99.2 Adenylylsulfate reductase activity is estimated from the difference in rate of reduction of ferricyanide (A4z0) in the assay described above, in the presence of sulfite with and without AMP. z2 Assays (3 ml in 1-cm cuvettes) contain 0.1 M Tris-HCl (pH 7.4), 1 mM potassium ferricyanide, 3 mM sodium sulfite, and cell-free extract. The rate of ferricyanide reduction due to AMP-independent sulfite oxidation is determined for a few minutes before 0.05 ml of AMP (0.75 mM) is added to the cuvette and the rate of ferricyanide reduction measured for a further 5-10 min. Any stimulation of the rate of ferricyanide reduction by AMP is a measure of sulfite oxidation that can be ascribed to APS reductase. Controls should omit sulfite or AMP. The sequence of additions of AMP, sulfite, and extract should be tested to ensure that results obtained are independent of the order of exposure of the enzymes to the substrates. 21 W.-P. Lu and D. P. Kelly, J. Gen. Microbiol. 130, 1683 (1984). 22 T. J. Bowen, F. C.Happold, and B. F. Taylor, Biochim. Biophys. Acta 118, 566 (1966).
[36]
OXIDATION OF THIOSULFATE AND POLYTHIONATES
509
Sulfur Oxygenase (Sulfur Dioxygenase, Sulfur:Oxygen Oxidoreductase, Sulfur-Oxidizing Enzyme): EC 1.13.11.18 S 8 + 8H20 + 802 ~- 8H2503
Sulfur oxygenase is assayed by determination of oxygen uptake and thiosulfate production in the presence of powdered sulfur and reduced glutathione. 23,24 Respirometric assays are conducted in an oxygen electrode cell using reaction mixtures (2 ml) containing 0.25 M Tris-HC1 (pH 7.8), sulfur (48 mg), catalase (0.25 mg), 2,2'-bipyridyl (0.1 mM), reduced glutathione (2.5 mM), and crude cell-free extract (0.05-0.25 ml). Oxygen uptake rates should be corrected for (low) chemical control oxidation rates. Activities measured at 30° with cell-free extracts of thiobacilli are generally low, requiring incubation periods of an hour or longer. The time course of thiosulfate formation in an identical assay mixture can be followed in small flasks shaken in air and sampled at intervals up to 3 hr. The samples (0.1 ml) are mixed with 0.1 ml of 1 M cadmium acetate and then assayed cyanolytically for thiosulfate (see [35] in this volume).
Glutathione-Independent Sulfur "Oxidase" Sulfur oxidation in cell-free extracts of some thiobacilli is seen in mixtures lacking glutathione, and is apparently due to an uncharacterized enzyme(s) that might link oxidation to electron transport, rather than to being an oxygenase25: S 8 + 24H20 ~- 8H2SO 3 + 32H + + 32eActivity can be measured in an oxygen electrode cell using an assay mixture (2 ml) containing 0.1 M Tris-HC1 (pH 8.0), powdered sulfur (30 mg), with and without Tween 80 (1%, w/v). Taylor z5 used about 25 mg of protein per assay and found that at least 15 mg was required to initiate the reaction.
Proteins A and B and Thiosulfate-Oxidizing Multienzyme System of Thiobacillus versutus An enzyme system located in the periplasmic space of T. uersutus is capable of the complete oxidation of thiosulfate to sulfate, without the intermediate formation or accumulation of polythionates, with the coupled reduction of several cytochromes. 26 This can be assayed only with a 23 I. Suzuki, Biochim. Biophys. Acta 1114, 359 (1965). 24 I. Suzuki and M. Silver, Biochim. Biophys. Acta 122, 22 (1966). 25 B° F. Taylor, Biochim. Biophys. Acta 170, 112 (1968). 26 W.-P. Lu, B. E. P. Swoboda, and D. P. Kelly, Biochim. Biophys. Acta 828, 116 (1985).
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M E T A B O L I S M OF P O L Y T H I O N A T E S
[371
complete system comprising at least two proteins (Mr 16,000 and 64,000) and two unusual c-type cytochromes. One protein (A) is a thiosulfatebinding enzyme and the other (B) contains an unusual binuclear manganese cluster. The mechanisms of action of these enzymes is unknown and the activity of the components of the system cannot be assayed independently. Their assay and properties are therefore not further described in this chapter, but the specialist reader is referred to the original literature. 26-31 27 W.-P. Lu and D. P. Kelly, J. Gen. Microbiol. 129, 1673 (1983). 28 W.-P. Lu and D. P. Kelly, J. Gen. Microbiol. 129, 3549 (1983). 29 W.-P. Lu and D. P. Kelly, Biochim. Biophys. Acta 765, 106 (1984). 3o W.-P. Lu, FEMS Microbiol. Lett. 34, 313 (1986). 31 R. Cammack, A. Chapman, W.-P. Lu, A. Karagouni, and D. P. Kelly, FEBS Lett. 253, 239 (1989).
[37] W h o l e - O r g a n i s m M e t h o d s for I n o r g a n i c Sulfur Oxidation b y C h e m o l i t h o t r o p h s a n d P h o t o l i t h o t r o p h s By DON P. KELLY and ANN P. WOOD Introduction This chapter describes some of the techniques that have been used in studies of sulfur compound oxidation and energy coupling using suspensions of intact organisms and for the separate identification and characterization of enzymes that are located in the periplasm, rather than the cytoplasm, of thiobacilli. In some cases the techniques are derived from those developed for studies on other microorganisms or using mitochondria, and are outlined primarily to demonstrate that the same principles apply to the less routinely studied lithotrophs. Some of the procedures provide examples of the use of the analytical methods described in [35] in this volume.
Demonstration of Periplasmic Location of Some Enzymes Involved in Sulfur Compound Oxidation in Thiobacilli Separation of the periplasmic fractions from both Thiobacillus versutus and Thiobacillus tepidarius has been achieved by methods based on a METHODS IN ENZYMOLOGY, VOL. 243
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