Dimethyl Sulfoxide → Leukotriene B4
Reaktionsgleichung
[CH3:1][CH2:2][CH2:3][CH2:4][CH2:5]/[CH:6]=[CH:7]\[CH2:8]/[CH:9]=[CH:10]\[CH:11]=[CH:12]\[CH:13]=[CH:14]\[C@@H:15]1[O:17][C@H:16]1[CH2:18][CH2:19][CH2:20][C:21]([OH:23])=[O:22].CS(C)=[O:26]>>[CH3:1][CH2:2][CH2:3][CH2:4][CH2:5]/[CH:6]=[CH:7]\[CH2:8][C@@H:9]([OH:26])/[CH:10]=[CH:11]/[CH:12]=[CH:13]/[CH:14]=[CH:15]\[C@@H:16]([OH:17])[CH2:18][CH2:19][CH2:20][C:21]([OH:23])=[O:22]Komponenten
3
Versuchsvorschrift
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. overnight 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. 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.
Aufarbeitung
- additionadded to the reaction mixture to a final concentration of 2 μM