MECHANISTM OF HYDROGEN SULFIDE FORMATION FROMI THIOSULFATE1

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1 MECHANISTM OF HYDROGEN SULFIDE FORMATION FROMI THIOSULFATE1 AKIRA KAJI2 AND W. D. McELROY McCollrnt-Pratt Institute and Department of Biology, Johns Hopkins University, Baltimore, Maryland Sulfidle formation by the reduction of sulfuir compounds has been observed in several microorganisms (Butlin, 1956). The earlier observation by Neuberg and Welde (1914) that yeast can produce hydrogen sulfide from thiosulfate has been studied in detail by Tanner (1918). Although the reduction of thiosulfate by extracts from Escherichia coli (Artman, 1956) an(d )esulphovibrio desulphuricans (Ishimoto and Koyama, 1957; Postgate, 1956) has been reported, the enzymatic mechanism of hydrogen sulfide formation has so far remained obscure. The present work has shown that a mechanism of hydrogen sulfide production by an enzyme preparation from yeast is dependent upon the interaction of thiosulfate with sulfhydryl compounds. The products of this enzymatic process are hycdrogen sulfide, sulfite, and oxidized suilfhydryls. The significance of the reaction in the biological synthesis of cysteine is discussed. MATERIALS AND METHODS Coenzyrnes and other materials. Glutatblione (GSH), sodium isocitrate, and glucose-6- plosphate were purchased from Schwartz Company. Baker reagent grade sodium thiosulfate was used. Homocysteine and eysteine were obtained from General Biochemical Company and reduced triphosphopyridine inucleotide (TPNH), and parachloromercuribenzoate from the Sigma Chemical Company. Colloidal sulfur was prepared by a mo(lification of the method described by Chitanii (1951). Seventy grams of concentrated sulfuric acid (specific gravity 1.84) was cooled to -1 C and 3 ml of a sodium thiosulfate solutioni containing 5 g of Na2S23.5H2 was added. To this mixture, 3 ml of distilled water was added. After the reaction was completed, the mixture was dialyzed against running tap water for 24 hr at room 1 Supported in part by Office of Naval Research and the Atomic Energy Commission. McColluin- Pratt Institute Contribution No Daizian Foundation for Medical Research Fellow. Received for publication October 17, 1958 temperature to remove sulfite and thiosulfate ions. A pale yellow supernatant fluid, obtained after removal of elemental sulfur by centrifugation, did not contain sulfite or thiosulfate as tested by fuchsin and iodometric titration, respectively. The amount of sulfur in this solution was measured gravimetrically after the water had been evaporated ip vacuo at room temperature. Preparation of enzyme. Fresh Fleishmann l)aker's yeast was dried in air for about two weeks at room temperature. One hundred grams of the dry yeast were suspended in 25 ml of 1 M dibasic sodium phosphate solution, and kept at room temperature for 3 hr with occasional stirring. The suspension was then centrifuged, the pellet reextracted with 1 ml of 1 M dibasic sodium phosphate and both extracts combined. In order to eliminate soluble cofactors, the extract was saturated with ammonium sulfate, the ph was adjusted to 7. by.7 M ammonium hydroxide and the resulting precipitate, after centrifugation, was dissolved in.2 M phosphate buffer (ph 7.). The protein was precipitated a second time by saturated ammonium sulfate and after dissolving in phosphate buffer was dialyzed against.5 M phosphate buffer, ph 7., at C for 24 hr. This fraction I had a specific activity of The enzyme responsible for catalyzing the direct reduction of thiosulfate was partially purified by ammonium sulfate fractionation of the crude preparation I. The preparation II obtained from the 3 to 6 per cent saturated ammonium sulfate fraction had a specific activity of 2.. The specific activity of fraction I1 was almost doubled by heating at 6 C for 5 min (ph 7.). After removing the precipitate by centrifugation, the supernatant fraction III had a specific activity of 37.. There was a 75 per cent loss of enzymatic activity after boiling preparation I for 5 min in 2 M phosphate buffer, and complete loss of activity after 3 min of boiling. Ashed residue of preparation I had no activity. Hydrogen sulfide determination. Hydrogen sulfide was measured by the method described by 63

2 1959] HYDROGEN SULFIDE FORMATION FROM THIOSULFATE 631 Delwiche (1951). Lead acetate dissolved in gum arabic solution was added to reaction mixtures at various time intervals and the colloidal lead sulfide formed was meastured by the increase in optical density at 49 m,, using a Beckman speetrophotometer (model B). Sidlftte determination. Sulfite was determined by decolorization of basic fuchsin solution, measured at 54 mnu. It was necessary to eliminate the hydrogen sulfide formed coneurrently with the sulfite, since it also decolorized the fuchsin. This was accomplished by adding 4 ml of zinc carbonate slurry (Kurtenacker, 1927) to 6.5 ml of the reaction mixture, followed by centrifugation. To 1 ml of distilled water were added 1 ml of the supernatant fluid, 1 ml of fuchsin solution (1 mg of basic fuchsin in 1 ml H2),.5 ml of 1 M phosphate buffer (ph 7.2), and 1 ml of parachloromercuribenzoate (1 umoles/ml). The final volume was adjusted to 25 ml with water. The parachloromercuribenzoate was added to avoid slight fuchsin decolorization by glutathione. A standard curve for fuchsin decolorization by sulfite was made by adding known amounts of sulfite to the enzyme reaction mixture lacking thiosulfate. Thiosulfate and sulfite solutions were standardized by iodometric titration (Hawk et al., 1954). Measurement of oxidized glutathione. Oxidized glutathione concentration was measured by reducing it with TPNH using the glutathione reductase present in fraction I. Utilization of TPNH was measured by the change in optical density at 34 m,u (Rall and Lehninger, 1952). The rate of reduced triphosphopyridine nucleotide disappearance during the first 15 min is linearly proportional to the amount of oxidized glutathione added to the reaction mixtures (between 4 X 1-2,umoles and 2 X 1-2,moles of oxidized glutathione in 3 ml of reaction mixture). Assay of enzymatic activity. The following reaction mixture was employed for thiosulfate reduction: To 5 ml of glutathione (1,umoles) were added.5 ml of 2 M phosphate buffer (ph 7.), 1 ml of thiosulfate (27,umoles), and.2 ml of enzyme solution. The ph of the reaction mixture was 6.9. The reaction was carried out aerobically at 35 C in a Thunberg tube. Hydrogen sulfide was absorbed by 2 ml of 2 M NaOH placed in the upper chamber of the Thunberg tube. After 3 min incubation, the NaOH solution in the upper chamber was tipped into the reaction inixture and the tube was placed in an ice-bath to prevent nonenzymatic production of hydrogen F.8 z LL.6 4 o--j C-) ~ TIME (MIN) Figure 1. Glutathione reductase and H2S formation. The complete system contained 3 mg of protein (fraction I), 5 MAmoles of oxidized glutathione,.2 mg of triphosphopyridine nucelotide (TPN), 135,moles of thiosulfate, and TPN reducing systems (3,moles of glucose-6-phosphate and 4,umoles of isocitrate) in 6.5 ml of.15 m phosphate buffer (ph 7.1). = Complete system; * = systems where one of the above components is missing, and complete system with boiled enzyme. Incubation was carried out at 37 C. An optical density change of.5 corresponds to 2. /Amoles of H2S produced in the reaction mixture. sulfide from glutathione. The stopper was removed and 4 ml of lead acetate solution containing gum arabic were added. A unit of enzyme activity is defined as the increase in optical density of.1 at 49 mu after 3 min incubation. The specific activity is equal to optical density change X 1 per mg protein. RESULTS Reaction between glutathione and thiosldfate. Preliminary experiments indicated that veast extract would catalyze a reaction betweein glutathione and thiosulfate to produce hydrogen suilfide. This reaction may be analogous to the nonenzymatic reaction between cysteine and thiosulfate to produce hydrogen sulfide (Steigmann, 1945). Continuous hydrogen sulfide production from thiosulfate will take place provided a system is present which is capable of maintaining glutathione. As shown in figurp 1, all of the com-

3 632 KAJI AND McELROY [VOL ~) >3.3 z LuJ 2 C) ~~~~~~~~ TIME (MIN) Figure 2. Time course of hydrogen sulfide production by the reaction between thiosulfate and glutathione. The reaction mixture contained 1,Amoles of glutathione, 18,umoles of thiosulfate, and enzyme (12 mg of fraction II) in 6.7 ml of.15 M phosphate buffer (ph 6.8). Incubation was carried out at 36 C. An optical density change of.5 corresponds to 2.,umoles of H2S produced in the reaction mixture. * = With enzyme; = with partially inactivated enzyme (5 min 1 C); A\ = with completely inactivated enzyme (3 min 1 C). ponents of the reaction mixture are necessary for hydrogen sulfide production. The complete reaction mixture contained TPNH generating systems, oxidized glutathione, thiosulfate, and yeast enzyme. In any of the reaction tubes where one of the above mentioned components was absent, there was no hydrogen sulfide production. The results in figure 2 demonstrate that the yeast enzyme enhances the rate of reaction between glutathione and thiosulfate. The decrease in hydrogen sulfide production after 9 min incubation at 35 C can be attributed to the presence of sulfide oxidase in the preparation (Ichihara and McElroy, unpublished data). The incubation of sulfide with the enzyme preparation resulted in the rapid disappearance of sulfide and the formation of thiosulfate. The inhibitory action of sulfite which is produced in the reaction must also be considered as contributing to the decrease in rate of hydrogen sulfide production. 6 PROTEIN CONC.- MG Figure S. Relationship between enzyme concentration and H2S production. The reaction mixture contained 1,umoles of glutathione, 27,umoles of thiosulfate, and 12 mg of the enzyme (fraction II) in 6.7 ml of.15 M phosphate buffer (ph 7.4). Incubation was carried out at 35 C for 3 min. An optical density change of.5 corresponds to 2.,umoles of H2S produced in the reaction mixture. * = with enzyme (fraction II); = with partially inactivated enzyme (5 min, 1 C). The rate of hydrogen sulfide production was proportional to the amount of protein added (figure 3). In addition, the effect of thiosulfate concentration on the rate of hydrogen sulfide production in the presence of 1,umoles of glutathione was studied, and the Michaelis-Menten constant (Kin) was calculated to be 6 X 13 M. Identification of sulfite as a product. Ishimoto and Koyama (1957) showed that during the production of hydrogen sulfide from thiosulfate, sulfite is concurrently produced. Since thiosulfate at high temperature can react nonenzymatically with glutathione to produce hydrogen sulfide and sulfite, this reaction was studied in detail. The results in table 1 show that hydrogen sulfide and sulfite are produced in almost equimolar amounts. The results suggest that two moles of glutathione react with one mole of thiosulfate to form one mole of sulfite, hydrogen sulfide, and oxidized glutathione.

4 1959] HYDROGEN SULFIDE FORMATION FROM THIOSULFATE 633 TABLE 1 Nonenzynmatic formation of sulfite and hydrogen sulfide from thiosulfate and glutathione at high temperatures 5 Temp S3- produced H2S Produced SO3-/H2S C J,moles IAmoles The reaction mixture contained 1,moles of glutathione, 27 jamoles of S 3 in 6.7 ml of.15 M phosphate buffer (ph 6.9). 4 3 _ ().3 p- -4 I- =L.2 z ( TIME (MIN) Figure 4. Time course of sulfite and H2S production by the enzymatic reaction between glutathione and thiosulfate. Reaction mixture contained 1,umoles of glutathione, 27 jumoles of thiosulfate, and 12 mg of protein (fraction II) in 6.7 ml of.15 M phosphate buffer. Final ph was 6.8. Incubation was carried out at 35 C. = Increase of sulfite measured at 54 m, by fuchsin decolorization. An optical density change of.4 corresponds to 7.14,smoles of sulfite produced in the reaction mixture. Under the conditions described in Methods the optical density of 5.8 was obtained for the blank where no sulfite is present. O = Parallel increase of H2S. An optical density change of.5 corresponds to 2.,moles of H2S produced in the reaction mixture. D rc m. z Formation of sulfite was also demonstrated to occur in the enzymatically catalyzed reaction. Figure 4 shows the time course of sulfite and hydrogen sulfide production. The amount of sul- (I) (D C) c-l n Figure 6. Competitive inhibition of thiosulfate reduction by sulfite. Reaction mixture contained 1,umoles of glutathione, 6 mg of enzyme (fraction I), varied amount of thiosulfate ranging from 27,moles to 27,umoles, and sulfite in total volume of 7.6 ml of.15 M phosphate buffer (ph 6.9). 1/S is 1/ml of the thiosulfate solution (25,umoles/ml) added to the reaction mixture. 1/V is 1 X 1/optical density change in 3 min. The incubation was carried out for 3 min at 35 C. = 26.7 X 1-2 jumoles of S3= (3.5 X 15 M). = 8.9 X 1-2 moles of SO3- (1.16 X 1- M). fite produced during the reaction was larger than the apparent hydrogen sulfide production. This was due to the action of hydrogen sulfide oxidase in the preparation as mentioned before. There was no disappearance of sulfite when it was incubated with the enzyme preparation. The incubation of the enzyme with either glutathione or thiosulfate alone did not lead to the production of hydrogen sulfide. Site of enzyme action. Since hydrogen sulfide production can take place slowly without the enzyme, the enhancement of its production from glutathione and thiosulfate by the enzyme could be a result of the catalytic removal of one of the products. This possibility was eliminated by the following experiments. Incubation of varied amounts of oxidized glutathione (from 2 to 1,umoles) at 35 C for 3 min with 6 mg enzyme S

5 634 KAJI AND McELROY [VOL. 77 in 7.6 ml of.15 M phosphate buffer (ph 6.9) did not result in a decrease of oxidized glutathione. Also, sulfite did not decrease on incubation with the enzyme. Moreover, it was found that sulfite is not bound by the protein in a detectable amount (using the fuchsin decolorization method). From these results it can be concluded that the enzyme catalyzes the reaction between thiosulfate and glutathione directly. The further TABLE 2 Effect of cysteine and homocysteine on H2S production front thiosulfate Optical SH Compound S23 Enzyme DCehnsty at 49 mpa ymoles Homocysteine 6.2 Homocysteine Homocysteine DL-Cysteine DL-Cysteine DL-Cysteine The rest of the reaction mixture is as given in the text. An optical density change of.5 corresponds to 2.,umoles of H2S produced in the reaction mixture..7 mg possibility remained that the enzyme catalyzes a reaction between oxidized glutathione and sulfite. Although the reaction between oxidized glutathione and sulfite took place, the enzyme did not increase the rate of the reaction. This nonenzymatic reaction is analogous to that between cystine and sulfite described by Clarke (1932). Studies of inhibitors. Artman (1956) showe(d that sulfite strongly inhibits hydrogen stulfide production from thiosulfate bv an extract of E. co/i. Sulfite wvas also found to inhibit thie reaction catalyzed by the yeast extract. As showni by the Lineweaver and Burk plot (figure 5) the inhibitor competes with thiosulfate. Sulfite in concentrations up to 1-3 M did not have an inhibitory effect on the nonenzymatic reaction between glutathione and thiosulfate at 75 C, ph 7., whereas 3.5 X 1-5 M S3- was inhibitory to the enzymatic reaction. Parachloromercuribenzoate (1.4 X 1-3 M) was not inhibitory. Also, preincubation of the enzyme with this concentration of parachloromercuribenzoate for 15 min at room temperature, ph 7., followed by dialysis against phosphate buffer (.5 M, ph 7.) for 24 hr at to 5 C did not cause loss of activity. Cyanide (1.4 X 1- M) at pll 6.8 caused only 17 per cent inhibition of the reaction. Versene did not inhibit the reaction. O z H.2..1r Figure 6. Effect of ph on the enzymatic and nonenzymatic production of H2S. The reaction mixture contained 1,umoles of glutathione, 27 j,moles of thiosulfate, and 12 mg of protein (fraction I) in 6.7 ml of buffer solution. Acetate buffer (.8 M), below ph 5.2; phosphate buffer (.8 M), between ph 6. and 7.2; tris(hydroxymethyl)aminomethane buffer (.8 M), between ph 7.2 and 9.; and carbonate buffer (.8 M), above ph 9.4, were used. The reaction was carried out at 35 C for 3 min. = Enzymatic reaction; * = nonenzymatic reaction. An optical density change of.5 corresponds to 2.,moles of H2S produced in the reaction mixture.

6 1959] HYDROGEN SULFIDE FORMATION FROM THIOSULFATE 635 Oxidized glutathione had a slight iiihibitory effect on the nonenzymatic reaction occurriing at 8 C. At 1-3 M oxidized glutathione, a 4 per cent depression in the rate of the reaction was observed. The effect of oxidized glutathione on the enzymatic reaction was of the same order of magnitude, and therefore may be due to nonenzymatic reversal of the reaction. En2ymatic specificity. In addition to gluitatione, homocysteine, and cysteine were foutnd to react with thiosulfate to produce hydrogen sillfide. The nonenzymatic reaction between cysteine and thiosulfate was much faster than that between glutathione and thiosulfate (table 2 an(d figure 2). In this experiment each reactioni miiixture containing 1,umoles of the sulfhydryl compound was incubated at 37 C, ph 6.9, for 3 min. Ascorbic acid was found to be unable to reduce thiosulfate even in the presence of thie enzyme. Effect of hydrogen ion concentration. Enzymatic catalysis of thiosulfate reduction was maximum at about ph 8.6 (figure 6). However, it is douibtful whether this is the true enzymatic ph optimum because glutathione is oxidized spontaneously at high ph values, thus decreasing the amount of available substrate. Thiosulfate is spontaneously decomposed to sulfite and elemental sulfur at the lower ph. Sulfur reacts spontaneously witlh glutathione to produce hydrogen sulfide. Because of this decomposition reaction at lower ph, the production of hydrogein sulfide by glutathione and thiosulfate is increased as shown in figure 6. DISCUSSION Since glutathione is very abundant in yeast, it seems very likely that it serves as the redtietanit in the enzymatic formation of hydrogen sulfide from thiosulfate. HowAever, other sulfhvdryl cointaining compounds such as cysteine and homocysteine will also function in the same capacity. Systems capable of reducing oxidized glutathione were found essential for the continuous reduction of thiosulfate to sulfide and sulfite. The coupling of yeast glutathione reductase to the TPNH-glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase systems was found to be satisfactory for this purpose. In the light of this finding, the report of Artman (1956) that pyruvate acts as the best hydrogen donor for sulfide production is understandable. Because of the presence of hydrogen sulfide oxidase and a rapid nonenzymatic formation of thiosulfate from hydrogen sulfide in the presence of sulfite, and oxidized glutathione, it was not possible to study quantitatively the effect of the enzyme on the reversibility of the reaction. Sulfite ion competitively inhibits the enzymatic formation of sulfide. This is similar to its effect on rhodanese (Sorbo, 1951) and suggests the mechanism for the reduction of thiosulfate by an enzyme (ENZ) as shown in schema 1. S ENZ + S-SO3= -> S ENZ S ENZ S-S-SO3- S-S-SO gltitathione ENZ + oxidized glutathione S + H2S + S3=. Schema 1 The strong inhibition by sulfite can be explained in a manner similar to that proposed by Fridovich and Handler (1956) for sulfite oxidation in which they propose that the initial step is a binding of the sulfite to the disulfide group of lipoate. In the nonenzymatic reaction between glutathione and thiosulfate two steps are suggested as shown in schema 2. S S3 -* S + S3= S + 2GSH - H2S + GSSG Over-all: 2GSH+S23 -> GSSG + S3= + H2S Schema 2 The increase in the rate of the nonenzymatic formation of hydrogen sulfide at low ph implies that the reaction is acid catalyzed. Assuming the reduction of S23= does proceed in the manner as shown in schema 2, a possibility that the enzyme increases the rate of only one or the other of the steps was tested. On the assumption that colloidal sulfur could participate in the reaction

7 636 KAJI AND McELROY [VOL. 77 the effect of the enzyme on the spontaneous reaction between sulfur and glutathione was studied. Varied amounts of colloidal sulfur were incubated with 1 j,moles of glutathione in the absence and the presence of fraction III at ph 6.3. The enzyme did not increase the rate of hydrogen sulfide production from colloidal sulfur and glutathione. If the reaction proceeds by these two steps, the enzyme must catalyze the first reaction. However, the incubation of thiosulfate and large amounts of enzyme under standard experimental conditions did not produce a detectable amount of free sulfite. From these experimental data, it is suggested that yeast enzyme catalyzes hydrogen sulfide production from thiosulfate and glutathione by enhancing the splitting of the S-S bond in thiosulfate only in the presence of SH-compounds. It is possible that the SH-compound removes the elemental sulfur as it is formed on the surface of the enzyme. Ishimoto and Koyama (1957) and Ishimoto et al. (1957) have suggested from studies on the mechanism of thiosulfate reduction in bacteria, that molecular hydrogen, activated by hydrogenase, was responsible for the reduction of cytochrome C3 which in turn reduced thiosulfate by means of an unknown intermediary carrier. It is possible that some SH-compound like glutathione plays the role of this intermediary electron carrier. However, from inhibitor studies no evidence was obtained for participation of metal ion in the reaction catalyzed by the yeast enzyme. Concerning the biological significance of hydrogen sulfide production in yeast, the recent report by Schlossmann and Lynen (1957) that cysteine is formed from hydrogen sulfide and L-serine in the presence of pyridoxal phosphate and an enzyme from yeast, is of considerable interest. In view of these experiments, the splitting of thiosulfate, followed by reduction by some SH-compound in the cell, could play an important role in over-all sulfur metabolism. However, it is also possible, as suggested by Shepherd (1956) that thiosulfate can be incorporated directly into organic sulfur before being split, thus by-passing the stage of hydrogen sulfide. ACKNOWLEDGMENTS The authors are greatly indebted to Dr. Ballentine for his interest and to Dr. Ichihara for assaying hydrogen sulfide oxidase activity in the yeast extract. SUMMARY A crude enzyme preparation from yeast which catalyzes the formation of hydrogen sulfide from thiosulfate and glutathione was studied. The protein fraction responsible for this catalysis has been purified nearly fourfold. Sulfite and sulfide are the equimolar products formed in the reaction. When the reaction is coupled to glutathione reductase, sulfide is produced continuously from thiosulfate. Sulfite was found to be a competitive inhibitor of the enzymatic reaction. Thiosulfate could reverse this inhibition. A mechanism similar to that proposed for rhodanese has been postulated for this reaction. The physiological significance of this hydrogen sulfide formation is discussed. REFERENCES ARTMAN, M The production of hiydrogen sulfide from thiosulphate by E. coli. J. Gen. Microbiol., 14, BUTLIN, K. R Formation enzymatiqiie de sulfure a partir de substrats mineraux par les microorganismes. Colloque sur la biochemie du soufre..ditions du Centre National de la Recherche Scientifique 13, Quai Anatole France, Paris. CHITANI, T Inorganic chemistry. Sangyo Tosho Co., Tokyo. CLARKE, H. T The action of sulfite upon cystine. J. Biol. Chem., 97, DELWICHE, E. A Activators for the cysteine desulfhydrase system of an Escherichia coli. J. Bacteriol., 62, FRIDOVICH, I. AND HANDLER, P The initial step in enzymatic sulfite oxidation. J. Biol. Chem., 223, HAWK, P. B., OSER, B. L., AND SUMMERSON, W. H Practical physiological cheinistry, 13th ed. Blakiston Co., New York. ISHIMOTO, M. AND KOYAMA, J Biochemical studies on sulfate reducing bacteria. VI. Separation of hydrogenase and thiosulfate reductase and partial purification of cytochrome and green pigment. J. Biochem. (Tokyo), 44, 233. ISHIMOTO, M., KOYAMA, J., YAGI, T., AND SHI- RAKI, M Biochemical studies on sulfate-reducing bacteria. VII. Purification of the cytochrome of sulfate reducing bacteria and its physiological role. J. Biochem. (Tokyo), 44, 413.

8 1959] HYDROGEN SULFIDE FORMATION FROM THIOSULFATE 637 KURTENACKER, Z Zur iodometrischen Analyse eines Gemenges von Sulfid, Sulfit, und Thiosulfat. Z. anorg. u. allgem. Chem., 161, NEUBERG, C. AND WELDE, E Phytochemische Reduktionen IX Die Umwandelung von Thiosulfat in Schwefelwasserstoff und Sulfit durch Hefen. Biochem. Z., 67, POSTGATE, J. R Cytochrome C3 and desulphoviridin; pigments of the anaerobe Desulphovibrio desulphuricans. J. Gen. Microbiol., 14, RALL, W. T. AND LEHNINGER, A. L Glutathione reductase of animal tissues. J. Biol. Chem., 194, SCHLOSSMANN, K. AND LYNEN, F Biosynthese des Cysteins aus Serin und Schwefelwasserstoff. Bioch. Z., 438, SHEPHERD, C. J Pathways of cysteine synthesis in Aspergillus nidulans. J. Gen. Microbiol., 15, SORBO, B On the active group in rhodanese. Acta. Chem. Scand., 5, STEIGMANN, A Reactions of sulphur with lipins, soaps, and cysteine. J. Soc. Chem. Ind. (London), 64, TANNER, F. W Studies on the bacterial metabolism of sulfur. J. Am. Chem. Soc., 4,