-SulfoBiotics-SulfoBiotics-SulfoBiotics-SulfoBioticsࡣࡌࡵ Sodium disulfide (Na2S2) Sodium trisulfide (Na2S3) Sodium tetrasulfide (Na2S4) Sodium Polysulfide Set Technical Manual ㏆ᖺࠊ◲㯤ཎᏊࡀ㐃⤖ࡋࡓࣃ࣮ࢫࣝࣇࢻࡸ࣏ࣜࢫࣝࣇࢻࡢࡼ࠺࡞ࢧࣝࣇ࢙ࣥ◲㯤 (Sulfane Sulfur) ࢆྵࡴศ Ꮚ✀ࡀ⏕యෆከࡃᏑᅾࡋ࡚࠸ࡿࡇࡀ᫂ࡽࡉࢀ࡚࠸ࡲࡍࠋࡇࡢࡼ࠺࡞ศᏊ✀ࡣࠊ◲Ỉ⣲ࡢ⏘⏕ࡸ㈓ⶶࠊ ᨺฟࡔࡅ࡛࡞ࡃࠊS- ࢫࣝࣇࣄࢻࣜࣝ࡞ࡢࢱࣥࣃࢡ㉁ෆࢳ࣮࢜ࣝࢆࢱ࣮ࢤࢵࢺࡋࡓࢩࢢࢼࣝఏ㐩ࡶ㛵ࡋ ࡚࠸ࡿࡇࡀ☜ㄆࡉࢀ࡚࠾ࡾࠊ㠀ᖖὀ┠ࡉࢀ࡚࠸ࡲࡍࠋࡲࡓࠊࣃ࣮ࢫࣝࣇࢻࡸ࣏ࣜࢫࣝࣇࢻࡣࠊ୍⯡ⓗ࡞ 㑏ඖ≀㉁࡛࠶ࡿࢩࢫࢸࣥࡸࢢࣝࢱࢳ࢜ࣥࡼࡾࡶ㑏ඖ⬟ࡀ㠀ᖖ㧗࠸ࡓࡵࠊ⏕యෆ࡛㧗ᗘ࡞ᢠ㓟≀㉁ࡋ࡚ᶵ ⬟ࡋ࡚࠸ࡿྍ⬟ᛶࡶ♧၀ࡉࢀ࡚࠸ࡲࡍࠋ Sodium polysulfide (Na 2 S n ) ࡣࠊࢧࣝࣇ࢙ࣥ◲㯤ࢆྵࡴศᏊࡋ࡚᭱ࡶ༢⣧࡞ᵓ㐀ࢆ᭷ࡍࡿࢧࣝࣇ࢙ࣥ◲㯤ࢻ ࢼ࣮࡛࠶ࡾࠊ Ỉ⁐ᾮ୰࡛ࡣ pH ᛂࡌ࡚✀ࠎࡢᵓ㐀ࢆ᭷ࡍࡿ Hydrogen polysulfide ࡋ࡚Ꮡᅾࡋࡲࡍࠋᮏヨ⸆㢮ࡣࠊ ࣃ࣮ࢫࣝࣇࢻࡸ࣏ࣜࢫࣝࣇࢻࡢࡼ࠺࡞ࢧࣝࣇ࢙ࣥ◲㯤 (Sulfane Sulfur) ࢆྵࡴศᏊ✀ࡢ⏕యෆᶵ⬟ࡢゎ᫂ࡸゎ ᯒ᭷⏝࡛ࡍࠋ ࠉ Fig. 1 ࢧࣝࣇ࢙ࣥ◲㯤 (Sulfane Sulfur) ࢆྵࡴศᏊ✀ Sodium disulfide (Na2S2) pKa1 = 5 pKa2 = 9.7 Sodium trisulfide (Na2S3) pKa1 = 4.2 pKa2 = 7.5 Sodium tetrasulfide (Na2S4) pKa1 = 3.8 pKa2 = 6.3 ͤ pKa ್ࡣୗグࡢᩥ⊩ࢆཧ↷ࡋ࡚࠸ࡲࡍࠋ J. Gun et al., “Electrospray Ionization Mass Spectrometric Analysis of Aqueous Polysulfide Solutions”, Microchim. Acta, 2004, 146, 229 Fig. 2 ෆᐜ ಖᏑ᮲௳ ⏝ୖࡢὀព ⏝᪉ἲ Sodium polysulfide (Na2Sn) ࡢᵓ㐀 pKa ್ SB02 -SulfoBiotics- Sodium disulfide (Na2S2) 㸸100 mg x 5 SB03 -SulfoBiotics- Sodium trisulfide (Na2S3) 㸸100 mg x 5 SB04 -SulfoBiotics- Sodium tetrasulfide (Na2S4)㸸100 mg x 5 SB13 -SulfoBiotics- Sodium Polysulfide Set 㸸Na2S2, Na2S3, Na2S4 ྛ 100 mg x 1 0 㹼 5Υ࡛ಖᏑࡋ࡚ࡃࡔࡉ࠸ࠋ ᮏヨ⸆㢮ࡣ㠀ᖖ྾‵ᛶࡀ㧗࠸ࡓࡵࠊศᐊ ᡠࡋ࡚ࡽࡈ⏝ࡃࡔࡉ࠸ࠋ 㛤ᑒᚋࡣࠊ❅⣲ᑒධࡋࠊ࡞ࡿࡃ᪩ࡃࡈ⏝ࡃࡔࡉ࠸ࠋ 1) Na2Sn ࢆ⛗㔞ࡋࠊ㉸⣧Ỉ㸦❅⣲ࣂࣈࣜࣥࢢฎ⌮῭㸧ࢆῧຍࡋࠊ100 mmol/l Na2Sn Ỉ⁐ᾮࢆㄪ〇ࡍࡿࠋ ࠉ㸦㸸100 mmol/l Na2Sn Ỉ⁐ᾮ㸹Na2S2 11 mg/ml, Na2S3 14.2 mg/ml, Na2S4 17.4 mg/ml㸧 2) ࡇࡢ⁐ᾮࢆᐇ㦂ᛂࡌ࡚㉸⣧Ỉ㸦❅⣲ࣂࣈࣜࣥࢢฎ⌮῭㸧࡛ᕼ㔘ࡋࠊࡈ⏝ࡃࡔࡉ࠸ࠋ ͤ㉸⣧Ỉࡣ⏝๓ 30 ศ㛫௨ୖࠊ❅⣲ࣂࣈࣜࣥࢢࢆ⾜ࡗ࡚ࡃࡔࡉ࠸ࠋ⁐Ꮡ㓟⣲ࡼࡗ࡚㓟ࡉࢀࡿྍ⬟ᛶࡀ࠶ࡾࡲࡍࠋ ͤㄪ〇ࡋࡓ Na2Sn Ỉ⁐ᾮࡣࠊಖᏑ࡛ࡁࡲࡏࢇࠋ⁐ᾮㄪ〇ᚋࠊࡍࡄࡈ⏝ࡃࡔࡉ࠸ࠋ Fig. 3 1 mmol/l Sodium polysulfide (Na2Sn) Ỉ⁐ᾮࡢ྾ࢫ࣌ࢡࢺࣝ SB02, SB03, SB04, SB13 Revised February 06, 2015 ᐇ㦂 1 -Hydrogen polysulfide ࡢ㑏ඖ⬟ ᐃ㸦WST-8 ἲ㸧1) 20 µmol/l WST-8 (PBS) 1ml 10 mmol/l Na2Sn Ỉ⁐ᾮ 10 µl ࢆῧຍࡋࡓ㸦⤊⃰ᗘ Na2Sn 100µmol/l㸧ࠋ 2) ᐊ ࡛ 30 ศ㛫㟼⨨ࡋࡓᚋࠊ96-well plate ࡢྛ࢙࢘ࣝ 100 µl ࡎࡘῧຍࡋࠊ࣐ࢡࣟࣉ࣮ࣞࢺ࣮ࣜࢲ࣮ࢆ⏝࠸࡚ 450 nm ྾ගᗘࢆ ᐃࡋࡓࠋ * WST-8 ࡣᑠ♫࡛㛤ⓎࡋࡓỈ⁐ᛶࢸࢺࣛࢰ࣒ࣜ࢘ሷ࡛ ࠶ࡾࠊ㑏ඖࡉࢀࡿࡇࡼࡗ࡚㯤Ⰽࡢ࣐࣍ࣝࢨࣥኚ ࡋࡲࡍࠋ Fig. 4 WST-8 㑏ඖࡼࡿⓎⰍ Fig. 5 ྛ✀㑏ඖ≀㉁ࡼࡗ࡚⏕ᡂࡉࢀࡓ WST-8 formazan ⏤᮶ࡢ 450nm ྾ගᗘ ୍⯡ⓗ࡞㑏ඖ≀㉁࡛࠶ࡿࢢࣝࢱࢳ࢜ࣥ (GSH) ࡸࢩࢫࢸࣥ㸦Cys㸧࡛ࡣࠊ㑏ඖᛂࡼࡿ WST-8 formazan ⏤᮶ࡢ ⓎⰍࡣほᐹࡉࢀ࡞ࡗࡓࡀࠊHydrogen polysulfide ࡛ࡣⓎⰍࡀほᐹࡉࢀࡓࠋ௨ୖࡢ⤖ᯝࡽࠊHydrogen polysulfide ࡣࠊࢢࣝࢱࢳ࢜ࣥ (GSH) ࡸࢩࢫࢸࣥ㸦Cys㸧ࡼࡾࡶ㠀ᖖ㧗࠸㑏ඖ⬟ࢆ᭷ࡍࡿࡇࡀ☜ㄆࡉࢀࡓࠋ ᐇ㦂 2 ࢧࣝࣇ࢙ࣥ◲㯤ࢻࢼ࣮ Na2S3) ῧຍࡼࡿ⣽⬊ෆࢧࣝࣇ࢙ࣥ◲㯤㔞ኚࡢゎᯒ 1) ⾑Ύධࡾᇵᆅ㸦DMEM㸧࡛ㄪ〇ࡋࡓ CHO ⣽⬊ᠱ⃮ᾮ ࢆ 96-well black clear bottom plate 104 cells/well ࡞ࡿ ࡼ࠺✀ࡋࠊCO2 ࣮ࣥ࢟ࣗ࣋ࢱ࣮㸦37Υ㸧ෆ୍࡛ᬌᇵ㣴ࡋࡓࠋ 2) ୖΎࢆ㝖ཤࡋࡓᚋࠊ↓⾑Ύᇵᆅ㸦DMEM㸧࡛⣽⬊ࢆ 2 ᅇὙίࡋࡓࠋ 3) 100 µmol/l ࢧࣝࣇ࢙ࣥ◲㯤ࢻࢼ࣮ Na2S3 (Sodium trisulfide) ࢆྵࡴ↓⾑Ύᇵᆅ㸦DMEM㸧100 µl ࢆྛ࢙࢘ࣝῧ ຍࡋࠊCO2 ࣮ࣥ࢟ࣗ࣋ࢱ࣮㸦37Υ㸧ෆ࡛ 15 ศ㛫㟼⨨ࡋࡓࠋ 4) ୖΎࢆ㝖ཤࡋࡓᚋࠊ↓⾑Ύᇵᆅ㸦DMEM㸧࡛⣽⬊ࢆ 2 ᅇὙίࡋࡓࠋ 5) 20 µmol/l SSP4 ( ↓⾑Ύᇵᆅ㸦DMEM100 µl ࢆ࢙࢘ࣝῧຍࡋࠊCO2 ࣮ࣥ࢟ࣗ࣋ࢱ࣮㸦37Υ㸧ෆ࡛ 15 ศ㛫 㟼⨨ࡋࡓࠋ 6) ୖΎࢆ㝖ཤࡋࡓᚋࠊPBS ࡛⣽⬊ࢆ 2 ᅇὙίࡋࡓࠋ 7) PBS 100 µl ࢆ࢙࢘ࣝῧຍࡋࡓᚋࠊ⺯ග㢧ᚤ㙾ࢆ⏝࠸࡚⣽⬊ࢆほᐹࡋࡓࠋ * SSP4 (SB10; -SulfoBiotics- SSP4) ࡣࢧࣝࣇ࢙ࣥ◲㯤ࢆ㑅ᢥⓗ᳨ฟࡍࡿࡓࡵࡢ⺯ගࣉ࣮ࣟࣈ࡛ࡍࠋ ᮍฎ⌮ࠉࠉࠉࠉࠉࠉࠉࠉࠉࠉࠉ100 µmol/l Na2S3 ฎ⌮ࠉ ࠉ㸦㟢ග㛫 1000 msec㸧 Na2S3㸦ࢧࣝࣇ࢙ࣥ◲㯤ࢻࢼ࣮㸧࡛ฎ⌮ࡋࡓ⣽⬊ෆᙉ࠸⺯ගࡀほᐹࡉࢀࡓࠋ ཧ⪃ᩥ⊩ 1) Y. Kimura, Y. Mikami, K. Osumi, M. Tsugane, J. Oka, and H. Kimura, FASEB J., 2013, 27, 2451. 2) S. Koike, Y. Ogasawara, N. Shibuya, H. Kimura, and K. Ishii, FEBS Lett., 2013, 587, 3548. 3) T. Ida, T. Sawa, H. Ihara. Y. Tsuchiya, Y. Watanabe, Y. Kumagai, M. Suematsu, H. Motohashi, S. Fujii, T. Matsunaga, M. Yamamoto, K. Ono, N. O. Devarie-Baez, M. Xian, J. M Fukuto, and T. Akaike, Proc. Natl. Acad. Sci. USA., 2014, 111, 7606. ࡈ㉁ၥ࣭ࡈせᮃࡣୗグࡲ࡛࠾ၥ࠸ྜࢃࡏࡃࡔࡉ࠸ࠋ 㸺㛤Ⓨඖ㸼 Dojindo Molecular Technologies, Inc. 30W Gude Dr, Suite 260, Rockville, Maryland, 20850 U.S.A. Tel: +1-301-987-2667, Fax: +1-301-987-2687 URL: www.dojindo.com 㸺ጤク〇㐀ඖ㸼 ᰴᘧ♫ ྠோᏛ◊✲ᡤ ⇃ᮏ┴ୖ┈ᇛ㒆┈ᇛ⏫⏣ཎ 2025-5 ࠛ 861-2202 Tel:096-286-1515 Fax:096-286-1525 URL:www.dojindo.co.jp/ ࢻ࣮ࢪ࣭࣮ࣥࢫࢺ ( ᮾி ) Tel:03-3578-9651 Fax:03-3578-9650 SB02, SB03, SB04, SB13
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