title: A simple semi-quantitative in vivo method using H₂S detection to monitor sulfide metabolizing enzymes.
abstract: Here we present a simple in vivo microtiter plate assay using lead acetate [Pb(OAc)2]-soaked filter paper to detect H2S released by Escherichia coli metabolizing cysteine. The released H2S precipitates as brown lead sulfide (PbS) on Pb(OAc)2 soaked filter paper. The PbS stain quantitated by ImageJ software is proportional to the amount of H2S released from the culture. Expression of recombinant Acidithiobacillus ferrooxidans sulfide:quinone oxidoreductase (SQR) converts the H2S to sulfur, resulting in less PbS formation. The in vivo H2S oxidation activity of SQR was calculated based on the density of the PbS stain formed by E. coli expressing SQR compared with cells harboring the empty vector pLM1. The results are consistent with the in vitro activity of SQR measured by decylubiquinone (DUQ) reduction. This assay can be applied to sulfide metabolizing enzymatic studies, mutant screening and high-throughput inhibitor screens.
authors.affiliation: Membrane Protein Disease Research Group, Department of Biochemistry, University of Alberta, Edmonton, Canada.
authors.forename: Yanfei
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Membrane Protein Disease Research Group, Department of Biochemistry, University of Alberta, Edmonton, Canada.
authors.forename: Joel H
authors.initials: JH
authors.lastname: Weiner
mesh_terms: D001681:Biological Assay / Q000379:methods*; D015153:Blotting, Western; D002138:Calibration; D011232:Chemical Precipitation; D004926:Escherichia coli / Q000201:enzymology*; D006862:Hydrogen Sulfide / Q000032:analysis*; D007854:Lead / Q000737:chemistry; D013440:Sulfides / Q000737:chemistry / Q000378:metabolism*; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D013440:Sulfides; D014451:Ubiquinone; C018391:lead sulfide; D007854:Lead; C060262:2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone; D006862:Hydrogen Sulfide
keywords: decylubiquinone; lead acetate; microtiter plate; sulfide:quinone oxidoreductase