4.3. Anti-urease Inhibitory Properties

All chemical reagents were obtained from commercial suppliers and used without further purification. Urease (Type III, from Canavalia ensiformis), urea, acetohydroxamic acid (AHA; standard urease inhibitor) were purchased from (Sigma Aldrich, Prague, Czech Republic).

Urease inhibitory activity was evaluated using a system pump-injector (Agilent 1200) coupled with a Sciex-3200QTRAP- hybrid triple quadrupole/linear ion trap mass spectrometer fitted with Electrospray Ionization (ESI). The system runs in flow injection analysis (FIA) mode without a HPLC column. The operational parameter settings were as follows: curtain gas (CUR), 25 psi; nebulizer gas (GS1), 50; auxiliary gas (GS2), 40; declustering Potential (DP), 15 V; ion spray voltage, −4000 V; turbo temperature (TEM), 450 °C. MS in positive ion mode was used in multiple reaction monitoring (MRM) analysis for detection and quantitation of urea with transition m/z 61→44 (Figure 6). 0.1% HCOOH and 1 mM HCOONH₄ were used as mobile phases with the flow rate set at 0.5 mL∙min⁻¹. The injection volume was 10 µL.

Multiple reaction monitoring (MRM) analysis for detection and quantitation of urea by triple quadrupole mass spectrometry coupled with Electrospray Ionization (ESI) in positive ion mode with transition m/z (61→44).

Several important experimental conditions were investigated and taken into consideration to optimize the efficacy of the ESI-MS-based assay analysis, such as the buffer concentration, the buffer pH, and the type of sample vials. Under the optimized experimental conditions, experiments were employed and aimed at evaluating urease assay conditions that would be compatible with ESI-MS. The enzymatic reaction took place in 1 mM HCOONH₄ buffer, which was found to be suitable (pH 7.5) containing 60.0 µg∙mL⁻¹ of urease, over a substrate (urea) concentrations ranging from 137.0 to 694.1 µmol∙L⁻¹ at room temperature (25 °C). Urea was chosen as a natural substrate for the enzymatic reaction. Urea concentrations were maintained at concentrations below 694.1 µmol∙L⁻¹ to avoid excess substrate inhibition. The concentrations of inhibitors (AHA = 13.2 µmol∙L⁻¹ and AEHS = 81 µg∙mL⁻¹) were used and were within the range of their effectiveness to inhibit urease activity. Enzymatic reaction was carried out by pre-incubating urease in 1 mM HCOONH₄ buffer with each inhibitor during 180 min to reach binding equilibrium followed by adding urea. The solutions were directly injected automatically into FIA system and the concentration changes of urea were monitored. For the analysis of the kinetics of substrate depletion by ESI-MS, areas (total counts) under peaks for substrate in the FIA record were integrated. Each measured sequence consists of five measurements. Briefly, to determine the repeatability of measurements, we performed multiple measurements of enzymatic reaction of the same sample. The precision of time course analysis was calculated as RSD (%) of multiple measured slopes (lower than 10%). The slopes represent rate constants, which are used for determination of enzyme activity and inhibition studies. For clarity of figures, multiple measurements have not been presented in Figure 2 and Figure 4. As shown in Figure 5, multiple measurements and evaluation of slopes are presented.

For measuring the rate constants for determining K_m and V_max, we set to each experiment different substrate concentrations. Furthermore, for screening inhibitors, we set to each experiment different inhibitor concentrations or different types of inhibitor. A calibration curve after each experiment was measured to correct instability of MS signal.