Search for Volatile Product of the Reaction between Hemoglobin and Dimethyl Trisulfide



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The goal of these experiments was to search for volatile products of the reaction between dimethyl trisulfide and oxyHb, and to test the hypothesis that this reaction would produce methane thiol as a product. The detection of volatile compounds such as methane thiol is challenging, because their signals are often masked by those of more abundant components of the complex matrix in which they reside. This challenge was overcome by using a dual chamber cell to separate the gas detection chamber from the reaction chamber. The dual chamber approach was tested in experiments where the reducing agent tris (2-carboxy ethyl)phosphine was used in place of hemoglobin. Specifically, dimethyl trisulfide was mixed with tris (2-carboxy ethyl)phosphine in the reaction chamber, and the volatile thiols produced in this reaction transferred through the headspace into the detection chamber, where they reacted with 5, 5-dithiobis(2-nitrobenzoic acid)) to form the yellow colored 5-thio-2-nitrobenzoic acid. The production of 5-thio-2-nitrobenzoic acid was followed by UV-vis absorption spectrophotometry.
Oxyhemoglobin was prepared by reacting sodium dithionite with methemoglobin (metHb), and was then isolated by gel filtration chromatography. When the isolated oxyHb was reacted with DMTS, no color change in the detection chamber 5, 5-dithiobis(2-nitrobenzoic acid)) solution was observed. This contrasted strongly with the proof-of-principle experiments, which showed strong color changes in the detection chamber following the addition of dimethyl trisulfide to tris (2-carboxy ethyl)phosphine. The headspace gases above the reactions of dimethyl trisulfide with tris (2-carboxy ethyl)phosphine, and dimethyl trisulfide with oxyHb were also analyzed with (gas chromatography-mass spectrometry) GC-MS. These experiments showed that dimethyl trisulfide was consumed, and methane thiol was produced when dimethyl trisulfide was mixed with tris (2-carboxy ethyl)phosphine; and that dimethyl trisulfide was consumed, but no methane thiol was produced when dimethyl trisulfide was mixed with oxy hemoglobin. The most important novel conclusions of these experiments are that methane thiol was not detected as a product of the reaction between dimethyl trisulfide and oxy hemoglobin, and that no new large peaks were observed in the total ion chromatograms of the gases sampled from the headspace above the reaction. These observations suggest that the sulfur containing product of this reaction is likely to be a molecule other than methane thiol, which has a lower volatility than methane thiol. Because of the toxicity of the species that might be generated in this work, appropriate safety precautions were incorporated into each experiment.



Hemoglobin, Methemoglobin, Methyl mercaptan, Methane thiol, Dimethyl trisulfide, Tris(2-carboxy ethyl) phosphine, 5,5-dithio-bis-(2-nitrobenzoic acid), GCMS, Head space sample