Enzymatic Synthesis and Analysis of Oxylipins

(13S)-HPOT was prepared from 0.5 mM linolenic acid (LnA) using 7 μg soybean LOX (L7395, Sigma-Aldrich) in 1 mL 50 mM sodium phosphate buffer (pH 7.0) for 10 min at room temperature. An assay of the coupled OsAOC-OsAOS1 reaction was conducted as follows. OsAOS1 and OsAOC(I) (total 5 μg combined) were mixed to achieve molar ratios from 1:0 to 1:20 and added to (13S)-HPOT prepared by soybean LOX, to reach a total reaction volume of 2 mL. Reaction products were extracted. cis-OPDA was separated by straight phase HPLC (SP-HPLC), and the stereochemistry of cis-OPDA was analyzed by chiral phase HPLC (CP-HPLC) as described previously (Yoeun et al., 2013 (link)). Alternatively, the reaction mixture was injection directly, without extraction onto reverse phase HPLC (RP-HPLC), and HPLC was developed in methanol: water: acetic acid (80:20:0.01). All HPLC analyses were performed with UV detection at 205, 220, and 234 nm for α-ketol, OPDA, and HPOT, respectively.