Polydimethylsiloxane permeability-based method for the continuous and specific detection of hydrogen sulfide.
Hydrogen sulfide (H(2)S) is known to play a physiological role in processes as diverse as vasodilation, maintenance of vascular tone, neurotransmission, and immune response. The multitude of physiological functions in which H(2)S is involved warrants the development of useful methods for its detection. Here, we introduce a simple and continuous H(2)S detection method that exploits the relatively high polydimethylsiloxane (PDMS) permeability of H(2)S in comparison to other thiols typically encountered in the cellular milieu. In this method, 96-well inserts constructed of PDMS act as an H(2)S-permeable membrane, eliminating nonspecific thiol detection. This design also makes it possible to use virtually any available thiol-specific probe such as Ellman's reagent which was used here to detect H(2)S once it crossed the PDMS membrane. Utilizing this method, a detection limit of 9.2 ± 1.9 ppb(m) (parts per billion (by mole) or ~0.51 μM in 1.6 mL of buffer) free H(2)S (detected as solution sulfide) was achieved. In addition, the assay was used to determine K(M) and V(max) for natural substrates of cystathionine γ-lyase (CSE), the main enzyme responsible for H(2)S production in peripheral tissues. The K(M) and V(max) of CSE for cysteine were 3.79 ± 2.07 mM and 0.37 ± 0.02 nmol H(2)S/min, respectively. K(M) and V(max) for homocysteine were 6.90 ± 1.78 mM and 1.10 ± 0.19 nmol H(2)S/min, respectively. In addition, the assay was used to examine the potential for a direct interaction of H(2)S and NO. The levels of detected H(2)S decreased in the presence of NO under normoxia but not under anoxia indicating that H(2)S does not react with NO but with N(2)O(3) likely formed in the hydrophobic environment of PDMS.
Department of Chemistry & Biochemistry, University of Windsor, Windsor, Ontario, Canada N9B 3P4.
Adam
A
Faccenda
Jingyuan
J
Wang
Bulent
B
Mutus
D003542:Cystathionine gamma-Lyase / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004129:Dimethylpolysiloxanes / Q000737:chemistry*; D006710:Homocysteine / Q000378:metabolism; D006801:Humans; D006862:Hydrogen Sulfide / Q000032:analysis* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D008567:Membranes, Artificial; D009569:Nitric Oxide / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D010539:Permeability; D015203:Reproducibility of Results; D013997:Time Factors
D004129:Dimethylpolysiloxanes; D008567:Membranes, Artificial; D006710:Homocysteine; D009569:Nitric Oxide; C013830:baysilon; D003542:Cystathionine gamma-Lyase; D003545:Cysteine; D010100:Oxygen; D006862:Hydrogen Sulfide