A sensitive method for reliable quantification of sulfane sulfur in

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A sensitive method for reliable quantification of sulfane sulfur in biological samples Mingxue Ran, Tianqi Wang, Ming Shao, Zhigang Chen, Huaiwei Liu, Yongzhen Xia, and Luying Xun Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02875 • Publication Date (Web): 22 Aug 2019 Downloaded from pubs.acs.org on August 26, 2019

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Analytical Chemistry

A sensitive method for reliable quantification of sulfane sulfur in biological samples Mingxue Ran1,2, Tianqi Wang1, Ming Shao3, Zhigang Chen1, Huaiwei Liu1, Yongzhen Xia1*, and Luying Xun1, 4* 1State

Key Laboratory of Microbial Technology, Shandong University, Qingdao, 266237, People’s Republic of China.

2Institute

of Marine Science and Technology, Shandong University, Qingdao 266237, People’s Republic of China

3School

of Life Science, Shandong University, Qingdao 266237, People’s Republic of China

4School

of Molecular Biosciences, Washington State University, Pullman, WA, 99164-7520, USA.

E-mail: [email protected] E-mail: [email protected] ABSTRACT: Sulfane sulfur has been recognized as a common cellular component, participating in regulating enzyme activities and signaling pathways. However, the quantification of total sulfane sulfur in biological samples is still a challenge. Here, we developed a method to address the need. All tested sulfane sulfur reacted with sulfite and quantitatively converted to thiosulfate when heated at 95oC in a solution of pH 9.5 for 10 min. The assay condition was also sufficient to convert total sulfane sulfur in biological samples to thiosulfate for further derivatization and quantification. We applied the method to detect sulfane sulfur contents at different growth phases of bacteria, yeast, mammalian cells, and zebrafish. Total sulfane sulfur contents in all of them increased in the early stage, kept at a steady state for a period and declined sharply in the late stage of the growth. Sulfane sulfur contents varied in different species. For Escherichia coli, growth media also affected the sulfane sulfur contents. Total sulfane sulfur contents from different organs of mouse and shrimp were also detected, varying from 1 to 10 nmol/mg protein. Thus, the new method is suitable for the quantification of total sulfane sulfur in biological samples.

H2S has been referred as the third gasotransmitter, following NO and CO. It plays important roles in vasorelaxation, cardioprotection, resistance to oxidative stress, neurotransmission, and anti-inflammatory action,1-3 likely via protein persulfidation, affecting enzyme activities and facilitating signaling.4-7 H2S cannot directly react with protein thiols, but sulfane sulfur can; therefore, it is increasingly accepted that sulfane sulfur, a product of H2S oxidation, is mainly responsible for causing protein persulfidation.8-11 Sulfane sulfur includes persulfide (RSSH), polysulfide (RSSnR, RSSnH, and HSnH, n  2), and elemental sulfur (S8). HSnH and S8 are often present in the environment 12, and the organic polysulfide and persulfide are found inside cells. 13, 14 Sulfane sulfur is more versatile than thiols because sulfane sulfur species have both nucleophilicity and electrophilicity, while thiols have only nucleophilicity.15 The sulfane sulfur pool is thought to be both a source and sink of H2S,16 as it can be reduced by thioredoxin and glutaredoxin to H2S,17 which can be oxidized by sulfide:quionone oxidoreductase back to sulfane sulfur.18, 19 Inside cells, sulfane sulfur and H2S often coexist, explaining why both H2S and sulfane sulfur are referred as the signaling molecules.20 There are newly developed methods for the detection of specific sulfane sulfur species. Polysulfide can be detected by several new methods. Monobromobimane (mBBr) reacts with polysulfide to form bimane adducts, which can be separated by HPLC and detected with a fluorescence detector.21, 22 Since mBBr has strong electrophilicity and may further electrophilically attack the middle sulfane sulfur in the adduct,13 -(4-hydroxyphenyl)ethyl iodoacetamide (HPEIAM) with mild electrophilicity is recommended when long

chain polysulfide species are detected because it does not break the initially formed adducts.14 Further, HPE-IAM’s hydroxyphenyl residue may stabilize the adducts.23 HSnH reacts with trifluoromethanesulfonate to form dimethylpolysulfide species, which can be detected by using HPLC, distinguishing polysulfide species with varying chain lengths (H2Sn, n=2-9).24 The total sulfane sulfur has traditionally been detected by using a cyanide (CN-) method. CN- reacts with sulfane sulfur to form thiocyanate (SCN-), which combines with Fe3+ to form Fe(SCN)63−; the latter has a red color and can be detected by using a spectrophotometer.25, 26 The low sensitivity of this method limits its application.27 Although a modified HPLC method is used for the separation of SCN- from other components in the sample with overlapping absorption, the C30 reverse phase column requires frequent modification with 5% PEG20000, which is inconvenient for analyzing a large sample set.28 A new method is developed to determine total sulfane sulfur in biological samples, in which sulfane sulfur is reduced by a reducing agent to H2S that is then detected by the methylene blue method.29 However, the method may overestimate the sulfane sulfur contents, as it has a false positive response with sulfur containing compounds, such as L-cysteine, 3-mercaptopyruvate, and glutathione.30 Proper control is required to reduce the overestimation. In addition, a number of sulfane sulfur-sensitive fluorescent dyes are used for evaluating sulfane sulfur in living cells or in vitro,31-34 and some are effectively applied to monitor changes of cellular sulfane sulfur contents.35-37 The fluorescent probe SSP4 is also applied to estimate sulfane sulfur in biological samples,30 and a smartphone devise combined with the fluorescent probe

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SSP5 is able to rapidly and conveniently detect sulfane sulfur.38 These fluorescent probes are good for relative comparisons and real-time monitoring,38 but not for quantification. Thus, the detection of total sulfane sulfur in biological samples is still a challenge. Sulfite has been used to measure HSnH and S8 in environmental samples. It reacts with sulfane sulfur to produce thiosulfate, which is detected via titration with iodine,39, 40 linear sweep voltammetry,41, 42 and square wave voltammetry.43 These detection methods are good enough for the detection of high concentrations of sulfane sulfur but not sensitive for the quantification of sulfane sulfur in complex biological samples. Here, we optimized the reaction condition of sulfite with sulfane sulfur and applied a sensitive method for thiosulfate detection; the method was reliable for the quantitation of total sulfane sulfur in biological samples, including bacteria, yeast, mammalian cells, and animal organs.

EXPERIMENTAL SECTION Bacteria, cells, animals, and chemicals. Escherichia coli MG1655, Staphylococcus sciuri Z8 and Saccharomyces cerevisiae BY4742 were used for sulfane sulfide analysis. E. coli and S. sciuri were grown in lysogeny broth (LB). S. cerevisiae was grown in yeast extract peptone dextrose medium (YPD). FHC (normal colonic mucosal cell line) and HCT116 (colorectal cancer cell lines) were purchased from ATCC (Manassas, Virginia, USA) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). Zebrafish (Danio rerio) was given by Prof. Deli Shi; shrimp (Fenneropenaeus chinensis) was given by Prof. Xianwei Wang; mouse (Mus muscculus) was given by Prof. Xiaoyang Sun. Sodium hydrosulfide (NaHS), sulfur element (S8), reduced glutathione (GSH), oxidized glutathione (GSSG), cysteine, cystine, sulfite, thiosulfate, tetrathionate, and bis[3(triethoxysilyl)propyl] tetrasulfide (Tsp-SSSS-Tsp, Tsp2S4) were purchased from Sigma-Aldrich (Shanghai). Dimethyl trisulfide (Me-SSS-Me, Me2S3) was purchased from the TCI Company (Shanghai). Preparation of reactive sulfane sulfur species. H2Sn was prepared through the reaction of S8 with NaHS by using a reported method.12 Glutathione persulfide (GSSH) was prepared via reacting GSH with element sulfur following the protocol of Liu et al,44 and the concentration was determined with a colorimetric method.45 Cysteine persulfide (Cys-SSH) was prepared by following a reported method.46 The elemental sulfur (S8) detection. The dissolved elemental sulfur (S8) was extracted with chloroform according to a published protocol,24 and analyzed by HPLC using a C18 reverse phase column (VP-ODS, 150×4 mm, Shimadzu). The column was maintained at 40oC and eluted with pure methanol at a flow rate of 1 mL/min. The UV detector (SPD-M20A) was set at 273 nm. The sulfite method for sulfane sulfur detection. The reaction buffer consisted of 50 mM Tris-HCl buffer (pH=9.5) containing 1% Triton X-100, 50 μM DTPA, and 1 mM sulfite. The control butter contained no sulfite. Samples were mixed with the reaction buffer and the control buffer, respectively, and incubated at 95oC for 10 min to convert sulfane sulfur to thiosulfate. 50 μL of the sample was mixed with 5 μL of 25 mM monobromobimane (mBBr) in acetonitrile and incubated in the dark at room temperature for 30 min to convert thiosulfate into thiosulfate-bimane. An equal volume of acetic

acid and acetonitrile mixture (v/v=1:9) was added to precipitate proteins that are not soluble in organic solvents. The precipitates and cell debris were removed via centrifugation at 16,000 × g for 3 min. The supernatant was analyzed via HPLC with a fluorescence detector as reported.18 The difference of the detected concentration of thiosulfate in the reaction buffer and the control buffer was used to represent the sulfane sulfur content in the samples. Intracellular sulfane sulfur detection in microorganisms. A colony of E. coli, S. sciuri, and S. cerevisiae was picked up and cultured in LB or YPD medium overnight. E. coli was also cultured in M9 medium, a chemically defined medium. 500 µL of cultured cells were transferred into 50 mL of fresh medium. The cells were collected at defined incubation time, washed and re-suspended in 50 mM Tris-HCl buffer (pH=7.4) at OD600 of 3. Cell pellets from 1 mL of the suspension were re-suspended in 100 μL of the reaction buffer or control buffer for sulfane sulfur detection. To minimize the decomposition and oxidation of sulfane sulfur, the samples were processed quickly and excessive wash was not recommended. Cells culture and intracellular sulfane sulfur detection. FHC and HCT116 were grown in RPMI 1640 medium supplemented with 10% FBS. The medium was repeatedly changed to supply nutrients for the cells. The sulfane sulfur in one cycle of medium exchange was measured at defined time intervals. Cells were treated with 2 mL trypsin (0.25%, SigmaAldrich) for 3 min to break the adhesion between cells. Then 2 mL RPMI 1640 medium with 10% FBS was added, and the released cells were transferred into Eppendorf tubes. The cells were washed twice with phosphate buffer saline (PBS). Half of the cells was immediately used for sulfane sulfur detection. The other half was disrupted with ultrasonication in PBS for protein quantification. The protein concentration in the cell extract was estimated by using the BCA method.47 The sulfane sulfur content per mg of protein was reported. The detection of intracellular sulfane sulfur in higher living organisms. The tissues or organs of zebrafish (Danio rerio), mouse (Mus muscculus), and Chinese white shrimp (Fenneropenaeus chinensis) were frozen in liquid nitrogen and grinded in a crucible. Half of the disrupted cells was used for sulfane sulfur detection. The other half was re-suspended in PBS and disrupted by sonication for protein quantification. The sulfane sulfur content per mg of protein was reported.

RESULTS AND DISCUSSION Optimizing the reaction condition to convert various sulfane sulfur species into thiosulfate in vitro. To determine whether cyanide or sulfite was a better reagent towards sulfane sulfur, we tested their reactivity towards S8 and GSSH in TrisHCl buffer of pH 7.4 and 9.5 at room temperature (Figure S1). Sulfite reacted with both S8 and GSSH much faster than cyanide did; both reagents reacted with S8 faster than with GSSH; both reactions were faster at pH 9.5 than at 7.4. When heated at 95oC for 10 min, the reaction of sulfite with GSSH was all completed at pH ranging from 7.4 to 10.5 (Figure 1A). Specifically, the reaction of GSSH with sulfite at 95oC was completed within 6 min at pH 7.4 and 3 min at pH 9.5 (Figure S2). For E. coli cells, heating at pH 9.5 generated the most thiosulfate (Figure 1B). Thus, the reaction condition was carried out in 50 mM Tris buffer pH 9.5 with heating at 95oC for 10 min for further testing.


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Analytical Chemistry reaction.

Figure 1. The reaction of sulfite with GSSH or E. coli for thiosulfate production. (A) The reaction mixture contained 100 μM GSSH and 1 mM sulfite in 50 mM Tris-HCl buffer with pH ranged from 7.4 to 10.5; it was incubated at 95oC for 10 min. (B) The E. coli MG1655 cells were collected, washed, and re-suspended in 50 mM Tris-HCl buffer containing 1 mM sulfite with pH ranged from 7.4 to 10.5. Thiosulfate was measured after the mixtures incubated at 95℃ for 10 min. All data are average of at least three samples with standard deviation (error bar). We tested whether the inclusion of chelating agents and detergents in the reaction buffer would facilitate the release of sulfane sulfur or the production of thiosulfate from whole cells. SDS and deoxycholic acid (NaDC) decreased the efficiency, while 50 μM DTPA with 1% Triton X-100 did not affect the thiosulfate production from the reaction with E. coli cells (Figure S3). Therefore, we finalized the reaction buffer as 50 mM Tris-HCl buffer pH 9.5 containing 50 μM DTPA, 1% Triton X-100, and 1 mM sulfite, and the control buffer without sulfite. Further, different sulfur compounds including elemental sulfur (S8), inorganic polysulfide (H2Sn), persulfide (GSSH and Cys-SSH), organic polysulfide (Me2S3 and Tsp2S4), organic thiols (GSH and Cys), organic disulfide (GSSG), and sulfide (H2S) were used to check the specificity of the reaction. The results showed that thiosulfate was formed only in the samples containing sulfane sulfur (Figure 2). All tested sulfane sulfur species were quantitatively transformed into thiosulfate in the reaction buffer incubated at 95oC for 10 min (Figure 2). Thus, all types of sulfane sulfur react with sulfite to quantitatively produce thiosulfate under the assay condition. A standard curve of thiosulfate revealed a detection limit of 200 nM by using HPLC with a fluorescence detector (Figure S4), indicating the potential sensitivity of the assay. Total sulfane sulfur in vivo was successfully converted into thiosulfate for quantification. Whether the complex composition of cell contents affected the reaction was also checked. A series of defined concentrations of S8 was added to E. coli cells extracts and then measured. The detected thiosulfate concentration correlated well with the amounts of S8 added (Figure 3). The results indicate that the cell contents do not affect the detection of sulfane sulfur. E. coli, S. sciuri, and S. cerevisiae were tested for sulfane sulfur contents. Cells were collected and re-suspended in the reaction buffer and the control buffer. The samples were either directly incubated at 95oC for 10 min or subjected to ultrasonication to break cells and then heated for 10 min. The formed thiosulfate showed no difference (Figure 4), indicating heating is sufficient without sonication for the releasing of sulfane sulfur, which is converted to thiosulfate during the

Figure 2. The reaction of sulfite with inorganic and organic sulfane sulfur. 600 μM H2Sn, 700 μM S8, 100 μM GSSH, 50 μM Cys-SSH, 100 μM Me2S3, 150 μM Tsp2S4, 50 μM S4O62-, 100 μM GSSG, 100 μM Cys, or 20 μM NaHS was added to the reaction buffer. The thiosulfate produced was analyzed by HPLC after the mixture was incubated at 95oC for 10 min. All data are average of at least three samples with standard deviation (error bar).

Figure 3. Intracellular substances did not interfere with the reaction of sulfite with sulfane sulfur. E. coli MG1655 cells were suspended in the reaction buffer to OD600 of 2. S8 ranged from 0 μM to 50 μM was added to the mixtures, and the samples were incubated at 95oC for 10 minutes. The thiosulfate produced was measured, and the intracellular sulfane sulfur was subtracted. All data are average of at least three samples with standard deviation (error bar). Cellular sulfane sulfur contents of microorganisms at different growth stages. We further detected the sulfane sulfur contents according to growth stages in liquid media for E. coli, S. sciuri, and S. cerevisiae. They showed a similar trend. The sulfane sulfur contents increased in the early logarithmic growth phase, kept for a steady state in the middle to late of the logarithmic phase, and decreased sharply in the late logarithmic phase or early stationary phage (Figure 5AD). The minimum and maximum of total sulfane sulfur were 47.77 ± 5.16 and 392.66 ± 6.18 nmol·mL-1·OD-1 for E. coli, 5.05 ± 0.03 and 153.82 ± 29.30 nmol·mL-1·OD-1 for S.



cerevisiae. The increase from low to high were 8.2, 30.4 folds, respectively. The maximum total sulfane sulfur of S. sciuri was 138.37 ± 0.44 nmol·mL-1·OD-1 and the minimum was close to 0 nmol·mL-1·OD-1. E. coli cells kept sulfane sulfur at relatively higher levels for a longer period in LB medium than in M9 medium (Figure 5 A&B). The results suggest that E. coli contains as high as about 400 μM sulfane sulfur in LB medium and 300 μM in M9 medium when using a reported conversion factor: one mL of cells at OD600nm of one was converted to one μL of cellular volume (https://bionumbers.hms.harvard.edu/search.aspx). The content is similar to that estimated in E. coli by a different method.48

mammalian cells and zebrafish at various growth phases. We checked the sulfane sulfur content of mammalian cells in one single batch of the nutrient fed process. The sulfane sulfur content increased in the early stage and decreased in the late stage (Figure 6A). The sulfane sulfur content of zebrafish was also detected during embryo development stage and for adult fish. The sulfane sulfur content reached the highest level in the 6th day right before hatching and decreased after growing for several weeks as adult fish (Figure 6B). These results indicate that the sulfane sulfur content zebrafish embryo is the highest right before hatching into small fish on day 6. When using a cellular protein content of 200 g/L,49 the estimated sulfane sulfur content was 3.6 mM. Further, we detected sulfane sulfur in different organs from mouse and Chinese white shrimp (Figure 6C&D). As expected, all samples had sulfane sulfur, but its concentrations varied from about 1 to 10 nmol/mg of protein in different organs. The sulfane sulfur in mouse kidney has been reported to be 400-500 nmol/g tissue.50 Since the cells usually contain 200-300 g/L protein,49 the calculated sulfane sulfur could be 22.5 nmol/mg protein in mouse kidney, consistent with our result. The sulfane sulfur in mouse heart has been determined by reducing sulfane sulfur back to sulfide before detection as about 20 nmol/mg protein.51 The results may be an overestimate, for the method also converts acid-labile sulfur into sulfide.52

Figure 4. Thiosulfate production from E. coli cells via heating alone or heating with ultrasonication. E. coli cells were collected, washed, and re-suspended in the reaction buffer and the control buffer. The cells were either disrupted via ultrasonication and then incubated at 95oC for 10 min or directly incubated at 95oC for 10 min for the production of thiosulfate. All data are average of at least three samples with standard deviation (error bar).

Figure 6. Sulfane sulfur content in higher living organisms. (A) The mammalian cells; (B) zebrafish; (C) organs of mouse; (D) organs of shrimp. The data were normalized with protein contents. All data are average of at least three samples with standard deviation (error bar).

Figure 5. Intracellular sulfane sulfur changed with growth period. Intracellular sulfane sulfur contents (■) and OD600 (●) were measured associated with growth phases. (A) E. coli was cultured in LB; (B) E. coli in M9; (C) S. sciuri in LB; and (D) S. cerevisiae in YPD. All data are average of at least three samples with standard deviation (error bar). A survey of sulfane sulfur content in higher living organisms. We further detected the sulfane sulfur contents in

Cyanide has been reported to react with inorganic polysulfide under alkaline conditions,26 which is consistent with our observation that sulfite also reacts faster with sulfane sulfur under alkaline conditions than neutral conditions (Figure S1A). Thus, both can be used to analyze total sulfane sulfur in biological samples. However, sulfite is likely a better choice because it is a stronger reagent towards sulfane sulfur (Figure S1) and because the produced thiosulfate can be derived and determined by using HPLC with a fluorescence detector. Interestingly, thiocyanate can also react with mBBr to form SCN-bimane, which is detected and confirmed by LC-


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Analytical Chemistry MS.24, 53 Further, cyanide is toxic, and it should be avoided if possible. Because of its stronger activity and less toxicity, sulfite is recommended, rather than cyanide, for the detection of total sulfane sulfur in biological samples.

CONCLUSIONS This method is simple and effective to quantify total sulfane sulfur in biological samples, including bacteria, yeast, mammalian cells, animal organs, and small fish. All contain sulfane sulfur, but the contents are different among the tested organisms. The sulfane sulfur contents in all tested species vary with growth phases (Figure 5A, C&D). In addition, the growth media may also affect cellular sulfane sulfur contents, as E. coli produces more sulfane sulfur in the complex LB medium than in the defined M9 medium (Figure 5A&B). Interestingly, cellular sulfane sulfur contents in E. coli, zebrafish embryo, and mouse organs are relatively high. We hope that the method will complement with existing methods for the detection of elemental sulfur S8,24 specific small organic sulfane sulfur,23, 54 and protein persulfidation.14 Collectively these methods will facilitate the study of sulfane sulfur biology.

ASSOCIATED CONTENT Supporting Information Figure S1. Reaction of cyanide or sulfite with sulfane sulfur; Figure S2. The reaction of GSSH and sulfite at 95°C; Figure S3. Optimization of cell lysate preparation; Figure S4. The standard curve of thiosulfate by HPLC analysis.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected] E-mail: [email protected]

ORCID Mingxue Ran: 0000-0002-5708-3826 Huaiwei Liu: 0000-0002-0483-5318 Yongzhen Xia: 0000-0001-9950-1910 Luying Xun: 0000-0002-5770-9016

Author Contributions Y. Xia and L. Xun designed the method; M. Ran, did most of the experiments; M. Shao prepared the zebrafish samples; T. Wang, Z. Chen, H. Liu contributed to the research.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The work was financially supported by grants from the National Natural Science Foundation of China (31870085, 91751207, 31770093), the National Key Research and Development Program of China (2016YFA0601103). We thank Prof. Deli Shi for zebrafish, Prof. Xianwei Wang for shrimps, and Prof. Xiaoyang Sun for mice.

REFERENCES 1. Kimura, H., Hydrogen sulfide: from brain to gut. Antioxid. Redox Signaling 2010, 12 (9), 1111-1123. 2. Gadalla, M. M.; Snyder, S. H., Hydrogen Sulfide as a Gasotransmitter. J. Neurochemistry 2010, 113 (1), 14-26.

3. Rui, W., Hydrogen sulfide: the third gasotransmitter in biology and medicine. Antioxid. Redox Signaling 2010, 12 (9), 1061-1064. 4. Csaba, S., Hydrogen sulphide and its therapeutic potential. Nat. Rev. Drug Discovery 2007, 6 (11), 917-935. 5. Ling, L.; Peter, R.; Moore, P. K., Hydrogen sulfide and cell signaling. Annu. Rev. Pharmacol. Toxicol. 2011, 51 (1), 169-87. 6. Sen, N.; Paul, B.; Gadalla, M.; Mustafa, A.; Sen, T.; Xu, R.; Kim, S.; Snyder, S., Hydrogen Sulfide-Linked Sulfhydration of NF-κB Mediates Its Antiapoptotic Actions. Mol. Cell 2012, 45 (1), 13-24. 7. Mustafa, A. K.; Gadalla, M. M.; Nilkantha, S.; Seyun, K.; Weitong, M.; Gazi, S. K.; Barrow, R. K.; Guangdong, Y.; Rui, W.; Snyder, S. H., H2S signals through protein S-sulfhydration. Sci. Signaling 2009, 2 (96), ra72. 8. Ono, K.; Akaike, T.; Sawa, T.; Kumagai, Y.; Wink, D. A.; Tantillo, D. J.; Hobbs, A. J.; Nagy, P.; Xian, M.; Lin, J., Redox chemistry and chemical biology of H2S, hydropersulfides, and derived species: implications of their possible biological activity and utility. Free Radical Biol. Med. 2014, 77 (77), 82-94. 9. Toohey, J. I.; Cooper, A. J. L., Thiosulfoxide (sulfane) sulfur: new chemistry and new regulatory roles in biology. Molecules 2014, 19 (8), 12789-12813. 10. Yuka, K.; Yoshinori, M.; Kimiko, O.; Mamiko, T.; JunIchiro, O.; Hideo, K., Polysulfides are possible H2S-derived signaling molecules in rat brain. FASEB J. 2013, 27 (6), 2451-2457. 11. Hideo, K., Hydrogen sulfide and polysulfides as signaling molecules. Proc Jpn Acad Ser B Phys Biol Sci 2015, 47 (4), S6-S6. 12. Kamyshny Jr, A.; Borkenstein, C. G.; Ferdelman, T. G., Protocol for quantitative detection of elemental sulfur and polysulfide zero ‐ valent sulfur distribution in natural aquatic samples. Geostand. Geoanal. Res. 2009, 33 (3), 415-435. 13. Ida, T.; Sawa, T.; Ihara, H.; Tsuchiya, Y.; Watanabe, Y.; Kumagai, Y.; Suematsu, M.; Motohashi, H.; Fujii, S.; Matsunaga, T., Reactive cysteine persulfides and S-polythiolation regulate oxidative stress and redox signaling. Proc. Natl. Acad. Sci. 2014, 111 (21), 76067611. 14. Akaike, T.; Ida, T.; Wei, F. Y.; Nishida, M.; Kumagai, Y.; Alam, M. M.; Ihara, H.; Sawa, T.; Matsunaga, T.; Kasamatsu, S., Cysteinyl-tRNA synthetase governs cysteine polysulfidation and mitochondrial bioenergetics. Nat. Commun. 2017, 8 (1), 1177. 15. Li, H.; Liu, H.; Chen, Z.; Zhao, R.; Wang, Q.; Ran, M.; Xia, Y.; Hu, X.; Liu, J.; Xian, M., Using resonance synchronous spectroscopy to characterize the reactivity and electrophilicity of biologically relevant sulfane sulfur. Redox Biol. 2019, 101179. 16. Olson, K. R.; Gao, Y.; Arif, F.; Arora, K.; Patel, S.; DeLeon, E. R.; Sutton, T. R.; Feelisch, M.; Cortese-Krott, M. M.; Straub, K. D., Metabolism of hydrogen sulfide (H2S) and production of reactive sulfur species (RSS) by superoxide dismutase. Redox Biol. 2018, 15, 74-85. 17. Dóka, É.; Pader, I.; Bíró, A.; Johansson, K.; Cheng, Q.; Ballagó, K.; Prigge, J. R.; Pastor-Flores, D.; Dick, T. P.; Schmidt, E. E., A novel persulfide detection method reveals protein persulfide-and polysulfide-reducing functions of thioredoxin and glutathione systems. Sci. Adv. 2016, 2 (1), e1500968. 18. Xin, Y.; Liu, H.; Cui, F.; Liu, H.; Xun, L., Recombinant Escherichia coli with sulfide:quinone oxidoreductase and persulfide dioxygenase rapidly oxidises sulfide to sulfite and thiosulfate via a new pathway. Environ. Microbiol. 2016, 18 (12), 5123-5136. 19. Li, K.; Xin, Y.; Xuan, G.; Zhao, R.; Liu, H.; Xia, Y.; Xun, L., Escherichia coli uses different enzymes to produce H2S and reactive sulfane sulfur from L-cysteine. Front. Microbiol. 2019, 10, 298. 20. Jacob, C.; Anwar, A.; Burkholz, T., Perspective on recent developments on sulfur-containing agents and hydrogen sulfide signaling. Planta Med. 2008, 74 (13), 1580-1592. 21. Koike, S.; Kawamura, K.; Kimura, Y.; Shibuya, N.; Kimura, H.; Ogasawara, Y., Analysis of endogenous H2S and H2Sn in mouse brain by high-performance liquid chromatography with fluorescence and tandem mass spectrometric detection. Free Radical Biol. Med. 2017, 113, 355-362. 22. Kimura, Y.; Koike, S.; Shibuya, N.; Lefer, D.; Ogasawara, Y.; Kimura, H., 3-Mercaptopyruvate sulfurtransferase produces potential redox regulators cysteine-and glutathione-persulfide (Cys-SSH and GSSH) together with signaling molecules H 2 S 2, H 2 S 3 and H 2 S. Sci. Rep. 2017, 7 (1), 10459. 23. Hamid, H. A.; Tanaka, A.; Ida, T.; Nishimura, A.; Matsunaga, T.; Fujii, S.; Morita, M.; Sawa, T.; Fukuto, J. M.; Nagy, P.,



Polysulfide stabilization by tyrosine and hydroxyphenyl-containing derivatives that is important for a reactive sulfur metabolomics analysis. Redox Biol. 2019, 21, 101096. 24. Kamyshny, A.; Goifman, A.; Gun, J.; Rizkov, D.; Lev, O., Equilibrium distribution of polysulfide ions in aqueous solutions at 25 C: a new approach for the study of polysulfides' equilibria. Environ. Sci. Technol. 2004, 38 (24), 6633-6644. 25. Karchmer, J. H., Analytical chemistry of sulfur and its compounds. 1970. 26. Luthy, R. G.; Bruce Jr, S. G., Kinetics of reaction of cyanide and reduced sulfur species in aqueous solution. Environ. Sci. Technol. 1979, 13 (12), 1481-1487. 27. Shinkai, Y.; Kumagai, Y., Sulfane Sulfur in Toxicology: A Novel Defense System Against Electrophilic Stress. Toxicol. Sci. 2019. 28. Kamyshny Jr, A., Improved cyanolysis protocol for detection of zero ‐valent sulfur in natural aquatic systems. Limnol. Oceanogr. : Methods 2009, 7 (6), 442-448. 29. Ikeda, M.; Ishima, Y.; Shibata, A.; Chuang, V. T.; Sawa, T.; Ihara, H.; Watanabe, H.; Xian, M.; Ouchi, Y.; Shimizu, T., Quantitative determination of polysulfide in albumins, plasma proteins and biological fluid samples using a novel combined assays approach. Anal. Chim. Acta. 2017, 969, 18-25. 30. Bibli, S.-I.; Luck, B.; Zukunft, S.; Wittig, J.; Chen, W.; Xian, M.; Papapetropoulos, A.; Hu, J.; Fleming, I., A selective and sensitive method for quantification of endogenous polysulfide production in biological samples. Redox Biol. 2018, 18, 295-304. 31. Huang, Y.; Yu, F.; Wang, J.; Chen, L., Near-Infrared Fluorescence Probe for in Situ Detection of Superoxide Anion and Hydrogen Polysulfides in Mitochondrial Oxidative Stress. Anal. Chem. 2016, 88 (7), 4122. 32. Min, G.; Fabiao, Y.; Hao, C.; Lingxin, C., Near-infrared fluorescent probe for imaging mitochondrial hydrogen polysulfides in living cells and in vivo. Anal. Chem. 2015, 87 (7), 3631-8. 33. Jiang, G.; Li, M.; Wen, Y.; Zeng, W.; Zhao, Q.; Chen, C.; Yuan, H.; Liu, C.; Liu, C., Visualization of Sulfane Sulfur in Plants with a Near-Infrared Fluorescent Probe. ACS Sens. 2019, 4 (2), 434-440. 34. Takano, Y.; Hanaoka, K.; Shimamoto, K.; Miyamoto, R.; Komatsu, T.; Ueno, T.; Terai, T.; Kimura, H.; Nagano, T.; Urano, Y., Development of a reversible fluorescent probe for reactive sulfur species, sulfane sulfur, and its biological application. Chem. Commun. 2017, 53 (6), 1064-1067. 35. Gao, M.; Wang, R.; Yu, F.; Chen, L., Evaluation of sulfane sulfur bioeffects via a mitochondria-targeting selenium-containing nearinfrared fluorescent probe. Biomaterials 2018, 160, 1-14. 36. Gao, M.; Wang, R.; Yu, F.; Li, B.; Chen, L., Imaging of intracellular sulfane sulfur expression changes under hypoxic stress via a selenium-containing near-infrared fluorescent probe. J. Mater. Chem. B 2018. 37. Min, G.; Rui, W.; Yu, F.; You, J.; Chen, L., Imaging and evaluation of sulfane sulfur in acute brain ischemia using a mitochondriatargeted near-infrared fluorescent probe. J. Mater. Chem. B 2018, 6 (17), 10.1039.C7TB03200E. 38. Neill, D. L.; Chang, Y.-C.; Chen, W.; Li, L.; Xian, M., A smartphone based device for the detection of sulfane sulfurs in biological systems. Sens. Actuators, B: Chemical 2019, 292, 263-269. 39. Szekeres, L., Analytical chemistry of the sulphur acids ☆ . Talanta 1974, 21 (1), 1-44.

40. Jorgensen, B. B.; Kuenen, J. G.; Cohen, Y., Microbial transformations of sulfur compounds in a stratified lake (Solar Lake, Sinai) 1. Limnol. Oceanogr. 1979, 24 (5), 799-822. 41. Luther III, G. W.; Giblin, A. E.; Varsolona, R., Polarographic analysis of sulfur species in marine porewaters 1. Limnol. Oceanogr. 1985, 30 (4), 727-736. 42. Cutter, G. A.; Velinsky, D. J., Temporal variations of sedimentary sulfur in a Delaware salt marsh. Mar. Chem. 1988, 23 (3), 311-327. 43. Luther III, G. W.; Church, T. M.; Powell, D., Sulfur speciation and sulfide oxidation in the water column of the Black Sea. Deep Sea Res. , Part A. 1991, 38, S1121-S1137. 44. Liu, H.; Xin, Y.; Xun, L., Distribution, diversity, and activities of sulfur dioxygenases in heterotrophic bacteria. Appl. Environ. Microbiol. 2014, 80 (5), 1799-1806. 45. Flavin, M., Microbial transsulfuration: the mechanism of an enzymatic disulfide elimination reaction. J. Biol. Chem. 1962, 237, 768777. 46. Akaike, T.; Ida, T.; Wei, F.-Y.; Nishida, M.; Kumagai, Y.; Alam, M. M.; Ihara, H.; Sawa, T.; Matsunaga, T.; Kasamatsu, S., Cysteinyl-tRNA synthetase governs cysteine polysulfidation and mitochondrial bioenergetics. Nat. Commun. 2017, 8 (1), 1177. 47. Gill, S. C.; Von Hippel, P. H., Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 1989, 182 (2), 319-326. 48. Hou, N.; Yan, Z.; Fan, K.; Li, H.; Zhao, R.; Xia, Y.; Xun, L.; Liu, H., OxyR senses sulfane sulfur and activates the genes for its removal in Escherichia coli. Redox Biol. 2019, 101293. 49. Wiе⌡niewski, J. R.; Hein, M. Y.; Cox, J. r.; Mann, M., A “Proteomic Ruler” for protein copy number and concentration estimation without spike-in standards. Mol. Cell. Proteomics 2014, 13 (12), 34973506. 50. Iciek, M. g.; Bilska-Wilkosz, A.; Górny, M.; SokołowskaJe˙zewicz, M.; Kowalczyk-Pachel, D., The effects of different garlicderived allyl sulfides on anaerobic sulfur metabolism in the mouse kidney. Antioxidants 2016, 5 (4), 46. 51. King, A. L.; Polhemus, D. J.; Bhushan, S.; Otsuka, H.; Kondo, K.; Nicholson, C. K.; Bradley, J. M.; Islam, K. N.; Calvert, J. W.; Tao, Y.-X., Hydrogen sulfide cytoprotective signaling is endothelial nitric oxide synthase-nitric oxide dependent. Proc. Natl. Acad. Sci. 2014, 111 (8), 3182-3187. 52. Shen, X.; Peter, E. A.; Bir, S.; Wang, R.; Kevil, C. G., Analytical measurement of discrete hydrogen sulfide pools in biological specimens. Free Radical Biol. Med. 2012, 52 (11-12), 2276-2283. 53. Bhandari, R. K.; Manandhar, E.; Oda, R. P.; Rockwood, G. A.; Logue, B. A., Simultaneous high-performance liquid chromatographytandem mass spectrometry (HPLC-MS-MS) analysis of cyanide and thiocyanate from swine plasma. Anal. Bioanal. Chem. 2014, 406 (3), 727734. 54. Bianco, C. L.; Akaike, T.; Ida, T.; Nagy, P.; Bogdandi, V.; Toscano, J. P.; Kumagai, Y.; Henderson, C. F.; Goddu, R. N.; Lin, J., The reaction of hydrogen sulfide with disulfides: formation of a stable trisulfide and implications for biological systems. B. J. Pharmacol. 2019, 176 (4), 671-683.


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A sensitive method for reliable quantification of sulfane sulfur in

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