Analysis of LOX and HPL activities at 234 nm by spectrophotometry. Total proteins were isolated from yeast cells expressing Nb-9-LOX (A), Cm-9/13-HPL (B), and Cl-13-HPL (C) which were harvested at time points corresponding to 0 (-■-), 4 (-●-), 8 (-▲-), and 24 (-♦-) hours after galactose induction. Nb-9-LOX activity was measured at pH 7.0 using linoleic acid as a substrate, Cm-9/13-HPL activity was measured at pH 7.0 using 9(S)-HPOD as a substrate, and Cl-13-HPL activity was measured at pH 6.0 using 13(S)-HPOD as a substrate. LOX activity was measured at room temperature by the formation of the conjugated diene at 234 nm, while HPL activity was measured by the decrease of A234 due to cleavage of the substrate. (D) Reaction of linoleic acid with yeast cell extract of Nb-9-LOX (-Δ-), followed by addition of yeast cell extract of Cl-13-HPL (-□-) or yeast cell extract of Cm-9/13-HPL (-○-) at 90 seconds (arrow).