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Using gas chromatography/isotope ratio mass spectrometry to determine the fractionation factor for H 2 production by hydrogenases
Author(s) -
Yang Hui,
Gandhi Hasand,
Shi Liang,
Kreuzer Helen W.,
Ostrom Nathaniel E.,
Hegg Eric L.
Publication year - 2011
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.5298
Subject(s) - chemistry , hydrogenase , fractionation , mass spectrometry , isotope fractionation , deuterium , isotope , desulfovibrio , chromatography , hydrogen , isotope ratio mass spectrometry , gas chromatography , kinetic isotope effect , analytical chemistry (journal) , sulfate , organic chemistry , physics , quantum mechanics
Hydrogenases catalyze the reversible formation of H 2 , and they are key enzymes in the biological cycling of H 2 . H isotopes have the potential to be a very useful tool in quantifying hydrogen ion trafficking in biological H 2 production processes, but there are several obstacles that have thus far limited the application of this tool. Here, we describe a new method that overcomes some of these barriers and is specifically designed to measure isotopic fractionation during enzyme‐catalyzed H 2 evolution. A key feature of this technique is that purified hydrogenases are employed, allowing precise control over the reaction conditions and therefore a high level of precision. In addition, a custom‐designed high‐throughput gas chromatograph/isotope ratio mass spectrometer is employed to measure the isotope ratio of the H 2 . Using our new approach, we determined that the fractionation factor for H 2 production by the [NiFe]‐hydrogenase from Desulfovibrio fructosovorans is 0.273 ± 0.006. This result indicates that, as expected, protons are highly favored over deuterium ions during H 2 evolution. Potential applications of this newly developed method are discussed. Copyright © 2011 John Wiley & Sons, Ltd.