FIGURE 2.
In vitro isotope-labeling analysis of the ACO-catalyzed reaction. Assessment of solvent back-exchange rate for RAL (A) and C10-apocarotenal (B). Authentic RAL or C10-apocarotenal was added to a buffered reaction system containing H218O (99% 18O) and purified ACO, and the mixture was incubated at 28 °C with 500 rpm shaking. Samples were collected at the indicated time points. Solvent back-exchange rates for RAL and C10-apocarotenal were quantified as the ion peak area ratios of the 16O- and 18O-labeled species. Note the increased exchange rates induced by addition of 1% (w/v) BSA and/or 5% (v/v) acetic acid. Error bars represent S.D. from duplicate measurements. Mass spectra for RAL (C) and C10-apocarotenal (D) generated under standard H216O/16O2 conditions. E and F, mass spectra for the apocarotenoid products generated in an H218O/16O2 environment showed isotope distributions similar to those of the control spectra shown in C and D, which demonstrated a lack of 18O incorporation from the labeled water into the products. G and H, by contrast, spectra for both apocarotenoids generated in an H216O/18O2 milieu showed a 2-Da shift in their isotope distribution patterns, attributable to 18O incorporation into both products. The data are quantitated in Table 2.