ABSTRACT
Hydrogen sulfide (H2S) is an important gasotransmitter in both animals and plants. Many physiological events, including responses to stress, have been suggested to involve H2S, at least in part. On the other hand, numerous responses have been reported following treatment with H2S, including changes in the levels of antioxidants and the activities of transcription factors. Therefore, it is important to understand and unravel the events that are taking place downstream of H2S in signaling pathways. H2S is known to interact with other reactive signaling molecules such as reactive oxygen species (ROS) and nitric oxide (NO). One of the mechanisms by which ROS and NO have effects in a cell is the modification of thiol groups on proteins, by oxidation or S-nitrosylation, respectively. Recently, it has been reported that H2S can also modify thiols. Here we report a method for the determination of thiol modifications on proteins following the treatment with biological samples with H2S donors. Here, the nematode Caenorhabditis elegans is used as a model system but this method can be used for samples from other animals or plants.
Subject(s)
Caenorhabditis elegans/drug effects , Helminth Proteins/isolation & purification , Hydrogen Sulfide/pharmacology , Protein Processing, Post-Translational , Sulfhydryl Compounds/metabolism , Animals , Behavior, Animal/drug effects , Caenorhabditis elegans/metabolism , Disulfides/chemistry , Disulfides/metabolism , Electrophoresis, Polyacrylamide Gel , Fluoresceins/chemistry , Helminth Proteins/chemistry , Helminth Proteins/metabolism , Hydrogen Peroxide/pharmacology , Hydrogen Sulfide/metabolism , Movement/drug effects , Nitric Oxide/metabolism , Oxidation-Reduction , Reactive Oxygen Species/metabolism , Signal Transduction , Staining and Labeling/methods , Sulfhydryl Compounds/chemistry , Sulfides/chemistry , Sulfides/pharmacologyABSTRACT
The binding of Cd2+, Zn2+, Cu2+, Ni2+, Co2+, Mn2+, and Mg2+ to apo, holo, reconstituted horse spleen ferritin (HoSF), and native holo HoSF with phosphate removed was measured by gel-exclusion chromatography. Three classes of strong binding interactions (Kd < 10(-7) M) with apo HoSF at pH 7.5 were found for the various M2+ studied: high stoichiometric binding (30-54 M2+/HoSF) for Cd2+, Zn2+, Cu2+, with two protons released per metal bound; intermediate binding (16 M2+/HoSF) for Ni2+ and Co2+, with one proton released per metal bound; and low levels of binding (2-12 M2+/HoSF) for Mn2+, Mg2+, and Fe2+, with < 0.5 protons released per metal bound. M2+ binding to apo HoSF was nearly abolished at pH 5.5, except for Fe2+ and Cu2+, which remained unaffected by pH alteration. Holo HoSF bound much higher levels of M2+, a result directly attributable to the presence of phosphate binding sites. This conclusion was confirmed by decreased binding of M2+ to HoSF reconstituted in the absence of phosphate and by native holo HoSF with phosphate chemically removed. The binding of Cd2+ to apo HoSF was 54 per HoSF, but in the presence of developing core, the amount bound decreased to about 30 Cd2+/HoSF. This result indicated that Cd2+ and developing core were competing for the same sites on the HoSF interior, suggesting that 24 of the Cd2+ were bound to the inside surface. No other M2+ studied bound to the interior of HoSF by this criterion. Several of the M2+ appeared to bind strongly to the phosphate-free mineral core surface in reconstituted HoSF.