反应详情

Immediately before the assay, LTA4 was diluted to a concentration of 10 μM in assay buffer (without DMSO) and added to the reaction mixture to a final concentration of 2 μM to initiate the enzyme reaction. After incubation for 120 sec at room temperature, the reaction was stopped by adding 2 volumes of chilled quenching buffer, containing acetonitril with 1% acetic acid and 225 nM LTB4-d4 (Biomol). The samples were then kept at 4° C. over night to complete protein precipitation and centrifuged for 15 min at 1800 g. LTB4 formed was measured by LC-MS/MS using LTB4-d4 as an internal standard and an external LTB4 standard (Biomol) as reference. Briefly, the analyte was separated from LTB4 isomers formed by spontaneous hydrolysis of LTA4 using isocratic elution (modified protocol from Mueller et al. (1996), J. Biol. Chem. 271, 24345-24348) on a HPLC system (Waters, Milford, Mass.) and analyzed on a tandem quadrupole mass spectrometer (Waters). MRM transitions followed on 2 channels were 335.2>195.3 (LTB4) and 339.2>197.3 (LTB4-d4). Based on the amounts of LTB4 found at each inhibitor concentration, a dose-response curve was fitted to the data and an IC50 value was calculated.

1. additionadded to the reaction mixture to a final concentration of 2 μM
2. customthe enzyme reaction
3. additionby adding 2 volumes
4. temperatureof chilled
5. customquenching buffer
6. additioncontaining acetonitril with 1% acetic acid and 225 nM LTB4-d4 (Biomol)
7. customThe samples were then kept at 4° C. over night to complete protein precipitation
8. waitcentrifuged for 15 min at 1800 g