Measuring Kinetic Isotope Effects in Enzyme Reactions Using Time-Resolved Electrospray Mass Spectrometry

2013 ◽  
Vol 85 (7) ◽  
pp. 3758-3764 ◽  
Author(s):  
Peter Liuni ◽  
Ekaterina Olkhov-Mitsel ◽  
Arturo Orellana ◽  
Derek J. Wilson
Keyword(s):  
Mass Spectrometry ◽  
Electrospray Mass Spectrometry ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Time Resolved ◽  
Enzyme Reactions ◽  
Kinetic Isotope

Activation of methane by gaseous platinum(II) ions PtX+ (X = H, Cl, Br, CHO)

2005 ◽  
Vol 83 (11) ◽  
pp. 1936-1940 ◽  
Author(s):  
Detlef Schröder ◽  
Helmut Schwarz
Keyword(s):  
Mass Spectrometry ◽  
Key Words ◽  
Electrospray Ionization ◽  
Gas Phase ◽  
Bond Activation ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Methane Activation ◽  
Gas Phase Reactions ◽  
Kinetic Isotope

The gas-phase reactions of methane with the platinum(II) ions PtX+ with X = H, Cl, Br, and CHO are studied by mass spectrometry. The PtX+ ions are generated by electrospray ionization of methanolic solutions of hexachloroplatinic acid and hexabromoplatinic acid, respectively. Small to moderate intramolecular kinetic isotope effects determined for the C—H(D) bond activation of CH2D2 suggest that the activation of methane by gaseous PtX+ cations is subject to thermochemical control by the product channels. In addition, the PtCl2+ cation is also able to activate methane, whereas PtCl3+ is unreactive under the conditions chosen. Key words: gas-phase reactions, mass spectrometry, methane activation, platinum bromide, platinum chloride.

Measurement of (carbon) kinetic isotope effect by Rayleigh fractionation using membrane inlet mass spectrometry for CO2-consuming reactions

2006 ◽  
Vol 33 (12) ◽  
pp. 1115 ◽  
Author(s):  
Dennis B. McNevin ◽  
Murray R. Badger ◽  
Heather J. Kane ◽  
Graham D. Farquhar
Keyword(s):  
Mass Spectrometry ◽  
Isotope Ratio ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Good Effect ◽  
Pep Carboxylase ◽  
Bisphosphate Carboxylase ◽  
Membrane Inlet Mass Spectrometry ◽  
Rayleigh Fractionation ◽  
Kinetic Isotope

Methods for determining carbon isotope discrimination, Δ, or kinetic isotope effects, α, for CO2-consuming enzymes have traditionally been cumbersome and time-consuming, requiring careful isolation of substrates and products and conversion of these to CO2 for measurement of isotope ratio by mass spectrometry (MS). An equation originally derived by Rayleigh in 1896 has been used more recently to good effect as it only requires measurement of substrate concentrations and isotope ratios. For carboxylation reactions such as those catalysed by d-ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO, EC 4.1.1.39) and PEP carboxylase (PEPC, EC 4.1.1.31), this has still required sampling of reactions at various states of completion and conversion of all inorganic carbon to CO2, as well as determining the amount of substrate consumed. We introduce a new method of membrane inlet MS which can be used to continuously monitor individual CO2 isotope concentrations, rather than isotope ratio. This enables the use of a simplified, new formula for calculating kinetic isotope effects, based on the assumptions underlying the original Rayleigh fractionation equation and given by: --> The combination of inlet membrane MS and this formula yields measurements of discrimination in less than 1 h. We validate our method against previously measured values of discrimination for PEP carboxylase and RuBisCO from several species.

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