Chemical Name:Copper(2+), [N-(3',6'-Dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen]-5-yl)-1,4,7,10-tetraazacyclododecane-1-acetamide-κN1,κN4,κN7,κN10]-Coordination Compound
Appearance: Brown to Greenish brown solid
Purity: ≧ 90.0 %
Storage Condition: Store in a cool dark place.
Shipping Condition: Ambient temperature
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-
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 Curve
To quantitate the amount of hydrogen sulfide in a sample, A standard
curve was derived from the serial dilutions of hydrogen sulfide donor,
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 Na
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 Cells
1) 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.