Product Description: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 nitric oxide and carbon monoxide. However, around 80% of the total sulfide exists as hydrogen sulfide anion (HS- ) under physiological condition, since the pKa is about 7. In addition, HS- easily converts to various biochemical molecules such as persulfides and polysulfides, which react with sulfhydryl moieties in a living body. -SulfoBiotics- HSip-1 is a novel fluorescent probe to detect H₂S selectively and it emits strong green fluorescence when it reacts with H₂S. -SulfoBiotics-HSip-1 DA is cell membrane permeable and it enables fluorescent imaging of intracellular H₂S.

Fig. 1 Chemical structure of HSip-1

Fig. 2 Excitation and emission spectra of HSip-1 reacted with H₂S(Em: 491nm/Ex: 516nm)

1. K. Sasakura, K. Hanaoka, N. Shibuya, Y. Mikami, Y. Kimura, T. Komatsu, T. Ueno, T. Terai, H. Kimura, and T. Nagano, “Development of a Highly Selective Fluorescence Probe for Hydrogen Sulfide”, J. Am. Chem. Soc., 2011, 133, 18003.

Quantification of Hydrogen Sulfide in HeLa cells:1. Preparation of Standard CurveTo quantitate the amount of hydrogen sulfide in a sample, A standard curve was derived from the serial dilutions of hydrogen sulfide donor, Sodium Sulfide.1) HSip-1 stock solution (10 mmol/l) was diluted with PBS to prepare 200 μmol/l HSip-1 working solution.2) Sodium Sulfide (-SulfoBiotics- Sodium Sulfide (Na₂S), 7.8 mg) were dissolved in 1 ml of de-oxygenated H₂O prepared by bubbling of nitrogen gas (100 mmol/l Na₂S solution).3) Na₂S solution (100 mmol/l, 20 μl) was added to 980 μl of de-oxygenated H₂O to prepare 2 mmol/l Na₂S solution.4) Na₂S solution (2 mmol/l,100 μl) was added to 900 μl of de-oxygenated H₂O to prepare 200 μmol/l Na₂S solution.5) Na₂S solution (200 μmol/l) was diluted with de-oxygenated H₂O to prepare various concentration of Na₂S solution by serial dilution (200, 100, 50, 25, 12.5, 6.3, 3.2, 0 μmol/l).6) HSip-1 working solution (200 μmol/l, 350 μl) was added to 300 μl of the Na₂S solutions and mixed using a vortex mixer.7) The solutions were incubated at room temperature for 30 minutes and 200 μl of the solution were transfered to each well (96-well plate).8) The fluorescence intensities were measured at 516 nm (λex=491 nm) with a microplate reader.

Fig. Fluorescence intensity changes at 516 nm with various concentrations of hydrogen sulfide.

2. Experimental Example with HeLa Cells1) Inoculate HeLa cells in 96-well microplate and incubate the cells in a CO₂ incubator overnight.2) Wash the cells with PBS buffer and remove the supernatant.3) Add Lysis buffer * 100 μl to a well and pipet it to lyse cells.4) Add HSip-1 working solution 100 μl to a well and incubate the cells at room temperature for 30 minutes.5) Measure the fluorescence intensity on a plate reader.( Ex: 491 nm, Em: 516 nm)* Lysis buffer: 6 mol/l Urea, 2% SDS, 150 mmol/l Tris-HCl (pH7.4)The hydrogen sulfide concentration was 3 to 9 μmol/l in HeLa cells. The concentration was obtained from the standard curve, according to the fluorescence intensity of the sample.