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.
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 (16)O and (18)O 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 O2-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.
Division of Plant Science, Research School of Biology, the Australian National University , Canberra ACT 0200, Australia.
Mun Hon
MH
Cheah
A Harvey
AH
Millar
Ruth C
RC
Myers
David A
DA
Day
Justine
J
Roth
Warwick
W
Hillier
Murray R
MR
Badger
D013058:Mass Spectrometry; D008567:Membranes, Artificial; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology; D009862:Online Systems; D010088:Oxidoreductases / Q000737:chemistry*; D010101:Oxygen Consumption / Q000502:physiology*; D010103:Oxygen Isotopes / Q000737:chemistry*; D010104:Oxygen Radioisotopes / Q000737:chemistry*; D010105:Oxygenases / Q000737:chemistry
D008567:Membranes, Artificial; D010103:Oxygen Isotopes; D010104:Oxygen Radioisotopes; D010088:Oxidoreductases; D010105:Oxygenases