Physical state of inhibitor fatty acids and linoleate solutions under lipoxygenase assay conditions

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Abstract
A variety of fatty acids, which are potential competitive inhibitors of soybean lipoxygenase (EC 1.13.11.12), give kinetically unstable mixtures in standard assay solutions containing linoleate (substrate). In the assay solution (pH 10, 0.1 M borate, 1.63% ethanol), each fatty acid by itself shows normal surface tension vs concentration behavior; but, despite a range of solubilization techniques in the presence of 10 μM or higher linoleate and low concentrations of these materials, irreproducible surface‐tension readings and inhibition kinetics results. This inhomogeneity (or kinetic instability) disappears as the concentration increases. Critical micelle concentration (CMC) values of mixtures are not additive, and binary mixture behavior depends on fatty acid structure. Several lines of observation, including CMC values and actual surface tension (γ) values for several systems, suggest premicellar heterodimer or higher mixed aggregate formation. Lipids with Ki significantly above the irreproducible surface‐tension range give good kinetic behavior, and Ki is reported. The results are in accord with earlier work on aspects of these systems. Complementary solution physical studies must be done for any kinetic (or specificity) determinations of enzymes using lipids.