Online Oxygen Kinetic Isotope Effects Using Membrane Inlet Mass Spectrometry Can Differentiate between Oxidases for Mechanistic Studies and Calculation of Their Contributions to Oxygen Consumption in Whole Tissues

Mun Cheah, A. Millar, Ruth Myers, David Day, Justine Roth, Warwick Hillier, Murray Badger

    Research output: Contribution to journalArticlepeer-review

    11 Citations (Scopus)

    Abstract

    The reduction chemistry of molecular oxygen underpins the energy metabolism of multicellular organisms, liberating free energy needed to catalyze a plethora of enzymatic reactions. Measuring the isotope signatures of 16O and 18O during O2 reduction can provide insights into both kinetic and equilibrium isotope effects. However, current methods to measure O2 isotope signatures are time-consuming and disruptive. This paper describes the application of membrane inlet mass spectrometry to determine the oxygen isotope discrimination of a range of O 2-consuming reactions, providing a rapid and convenient method for determining these values. A survey of oxygenase and oxidase reactions provides new insights into previously uncharacterized amino acid oxidase enzymes. Liquid and gas phase measurements show the ease of assays using this approach for purified enzymes, biological extracts and intact tissues.

    Original languageEnglish
    Pages (from-to)5171-5178
    Number of pages8
    JournalAnalytical Chemistry
    Volume86
    Issue number10
    DOIs
    Publication statusPublished - 2014

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