-SulfoBiotics- Sodium sulfide (Na2S)SB01 Product Manual

It has been recognized that hydrogen sulfide (H₂S) has an important role as a physiological active substance for vasodilation, cytoprotection, and modulation of insulin secretion. H₂S is considered as a gaseous molecule such as NO and CO. However, around 80% of the total sulfide exists as hydrogen sulfide anion (HS^-) under physiological condition, since the pKa is about 7 (Fig. 1). In addition, H₂S easily converts to various biochemical molecules such as persulfides and polysulfides, which react with sulfhydryl moieties in a living body. The functional mechanism of H₂S has not been well understood.Sodium sulfide (Na₂S) has been widely used as a H₂S donor. Na₂S is readly decomposed and release H₂S when Na₂S is dissolved in H₂O.

Fig. 1 Physiological functions of hydrogen sulfide

-SulfoBiotics- Sodium sulfide (Na2S)

100 mg x 5

Store at 0-5 °C

• Use the reagent after bringing to room temperature, because it is moisture sensitive.
• Use up the reagent soon after opening.

1. Dissolve 7.8 mg of Na₂S in 1 ml of deionized H₂O under nitrogen gas (100 mmol/l Na₂S solution).
2. Dilute the 100 mmol/l Na₂S aqueous solution to an appropriate concentration depending on your experiment.

• Purge deionized H₂O with nitrogen gas for longer than 30 minutes to prevent the oxidation.
• Use the aqueous solution as soon as prepared. The solution is not stable enough to store.

- Detection of hydrogen sulfide by methylene blue method -

1. 20 µl of 100 mmol/l Na₂S aqueous solution was added to 980 µl of deionized H₂O to prepare 2 mmol/l Na₂S solution.
2. 100 μl of 2 mmol/l Na₂S solution was added to 900 µl of deionized H₂O to prepare 200 µmol/l Na₂S solution.
3. 200 µmol/l Na₂S solution was diluted with deionized H₂O to prepare various concentration of Na₂S solution by serial dilution (200, 100, 50, 25, 12.5, 6.3, 3.2, 0 µmol/l).
4. 300 μl of 1% zinc acetate solution , 50 μl of 20 mmol/l N,N-Dimethyl-p-phenylenediammonium (7.2 mol/l HCl) solution and 50 μl of 30 mmol/l FeCl_³ (1.2 mol/l HCl) solution were added to 250 μl of the Na₂S solutions and mixed using a vortex.
5. The solutions were incubated at room temperature for 30 minutes and transfered 200 µl of the solution to each well (96-well plate).
6. Measure the absorbance at 650 nm using a microplate reader (Fig. 2).

Fig. 2 Absorbance change at 650 nm depending on the concentration of hydrogen sulfide

1. R. Greiner, Z. Palinkas, K. Basell, D. Becher, H. Antelmann, P. Nagy and T. P. Dick, “Polysulfides link H₂S to protein thiol oxidation”, Antioxid. Redox Signal., 2013, 19, 1749.
2. N. S. Lawrence, J. Davis and R. G. Compton, “Analytical strategies for the detection of sulfide: a review”, Talanta, 2000, 52, 771.