Characterization of the substrate aggregation state in 5-lipoxygenase oxidation of linoleic acid

5-lipoxygenase ( EC 1.13.11.12 ) oxidizes polyunsaturated fatty acids by molecular oxygen. The enzyme acts in close contact with the cell membranes, which main components are ionic and non-ionic lipids. In order to investigate the kinetic parameters of 5-lipoxygenase reaction in vitro, extremely hydrophobic fatty acid substrate ( linoleic acid ) should be solubilized in the reaction mixture. We used Lubrol PX («Sigma» Chem. Co), as a non-ionic detergent consisted of oligoethylene glycol and fatty alcohol. Linoleic acid and Lubrol PX formed mixed micelles thus solubilizing the fatty acid substrate in a buffer with appropriate pH. We have studied the sizes and shapes of mixed micelles Lubrol PX / linoleic acid (aggregates type 1 ) and Lubrol PX / linoleic acid / SDS ( aggregates type 2; SDS was an effective activator of potato tuber 5-lipoxygenase) by means of gel-filtration and laser light scattering techniques. The parameters under investigation were molecular weights, Stocks radii and shapes of the mixed micelles. The average molecular weights and Stocks radii of the mixed micelles type 1 determined by mean of gel-filtration on Sephadex G-200 were 95 142 ± 5184 Da and 3,45 ± 0,11 nm, respectively. The same parameters for the mixed micelles type 2 were 73 694 ± 893 Da and 3,02 ± 0,02 nm, respectively. The strong similarity in physicochemical parameters for both types of mixed micelles indicated that SDS did not influence the size and shape of mixed micelles of Lubrol PX and linoleic acid. The activatory action of SDS on potato tuber lipoxygenase may be a result of electrostatic effect or direct participation of SDS in enzymatic catalysis. The laser light scattering technique allowed to determine two main fraction of particles in type 1 system with hydrodynamic diameters 2,6 and 5,7 nm and relative contribution to light scattering 13 and 87%, respectively. The particles with d=5,7 nm were interpreted as the mixed micelles. The particles with d=2,6 nm were interpreted as isolated molecules of Lubrol PX, linoleic acid and (or) their premicellar aggregates. The data obtained are to be used in creation of reliable physical and mathematical models of 5-lipoxygenase.