The assay of CA activity by MIMS has several advantages compared to other techniques: it is rapid and accurate over a wide temperature ranges, but a unique feature is that enzymatic activity is obtained at chemical equilibrium—i.e., under conditions of equilibrated CO2 and HCO3 − concentration. Other CA assays in contrast, using 14C labeling or pH transients, are reliant on rapid changes in the equilibrium that are slowed on ice and are not obtained at chemical equilibrium. The principle of the CA-MIMS assay is based upon

isotopic exchange of 18O-label between HCO3 − on selleck screening library one side of Eq. 9 and CO2 and water on the other side of the reaction. The MIMS assay monitors the [CO2] in solution, and thus provides a continuous real-time determination of one half of the reaction (Gerster 1971; Tu and Silverman 1975; Silverman 1982). As the isotopic approach deals with slow isotopic exchange reactions, it may be www.selleckchem.com/products/VX-680(MK-0457).html followed accurately for tens of minutes timescale. In practice, the MIMS assay is primed by the initial

addition of a known amount of 18O-hydrogencarbonate from a 200–500 mM stock.5 The assay is best performed with 13C-labeled Na-hydrogencarbonate as backgrounds are small, but can also be performed with 12C material if needed. The peaks of 13CO2 are then followed at m/z = 49, 47 and 45 for the 13C18,18O2, 13C16,18O2, and 13C16,16O2, respectively (Silverman 1982; Badger and Price 1989), as shown in Fig. 5a. After injection of hydrogencarbonate a rapid initial increase at m/z = 49, representing the initial short chemical equilibration between aqueous H13C18O3 − and gaseous 13C18O2 is Dolutegravir price observed (please notice the log scale on the time axis). This is followed by phases of isotopic equilibration with the eventual formation of 13C16O2 as the m/z = 45 species. Water provides the final sink for the 18O re-distribution and undergoes with time a gradual enrichment above natural abundance (Hillier et al. 2006; McConnell et al. 2007). Fig. 5 This assay for carbonic anhydrase activity of photosystem II samples shows the distribution of 13CO2

species following the injection of 50 mM H13C18O3 − into the liquid sample in the MIMS-cuvette. The experimental data (solid lines) were used to derive fitted amplitudes (dashes) at m/z = 49 (blue); m/z = 47 (red); m/z = 45 (green) and are this website plotted on a log time scales. A second plot to the right (B) gives the log of the 18O enrichment (also termed 18α) according to Eq. 5. For more details see (Hillier et al. 2006; McConnell et al. 2007) It is also possible to express isotopic exchange more qualitatively as the change in 18O enrichment (18α) as given by Eq. 6. When the enrichment is plotted as the natural log(18α) for CO2 versus time (Mills and Urey 1940) the slope of the line gives a measure of the pseudo first-order rate constant for hydration of CO2 by the CA reaction, see Fig. 5b.