The capability of IMS for the determination of kinetics in complex enzyme systems with reduced vmax values has been demonstrated with the example of diamine oxidase (DAO). Michaelis-Menten and Lineweaver-Burk plots were obtained for the enzyme catalyzed putrescine oxidation and calculations of the kinetic parameters have been performed and compared with previously published values. The IMS procedure provided a limit of detection of 200 pg mL-1 for putrescine, a limit of quantification of 667 pg mL -1, a precision of 5.9%, and an analysis frequency of 40 s, which are analytical characteristics appropriate to perform label-free enzyme studies. Additionally, pseudo-competitive inhibition of the putrescine oxidation due to other diamines binding to the free enzyme has been evaluated. Moreover, the mass-mobility correlation curve of diamines based on the calculated mobilities of the studied analytes and data previously reported for cadaverine, serotonin and tryptamine was modelled. The analytical method could be used as an additional complementary tool for the existing drug discovery method, for the search of DAO activators; molecules that displace the substrate from the second site of DAO, but does not interfere with the catalytic function of the first site. © The Royal Society of Chemistry 2012.
|Publication status||Published - 21 Dec 2012|