An octaethylene glycol monododecyl ether-based mixed micellar assay for lipoxygenase acting at neutral pH.
Using the detergent octaethylene glycol monododecyl ether (C12E8), a spectrophotometric mixed micellar assay for lipoxygenas (LOX) activity was designed. Potato LOX was able to use the linoleic acid (LA) solubilized in C12E8 micelles, displaying the characteristic induction period of LOX-catalyzed LA peroxidation. In the mixed micellar system, LOX responds to the LA surface concentration expressed as mol% (=[lipid]*100/([detergent]-cmc)) and not to the molarity of the LA. For both potato and soybean LOX, Vmax was independent of the mixed micelle concentration, while Km was independent as well when expressed as mol% but was dependent on C12E8 concentration when expressed in molar. In mixed micelles, H2O2 shortened the induction period, while 13-hydroperoxylinoleic acid and t-butyl hydroperoxide completely removed it. C12E8/LA proved to be a reliable system for assaying LOX activity at pH values around neutrality. Like Tween 20, this system avoided the turbidity problems arising from the protonated fatty acid and did not interfere with the uv-absorption band of the hydroperoxide product. However, this system is superior to the commonly used Tween 20 because it permits investigation of the lipid requirements of LOX since the concentration-independent Km can be determined both in mol% and as the absolute number of lipids per micelle. In addition, the detergent did not affect the enzyme through any side effects.
López-Nicolás
J M
JM
Departamento de Bioquímica y Biología Molecular A, Universidad de Murcia, Spain.
Bru
R
R
Sánchez-Ferrer
A
A
García-Carmona
F
F
D003902:Detergents; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019787:Linoleic Acid; D008041:Linoleic Acids; D008084:Lipoxygenase / Q000032:analysis* / Q000378:metabolism*; D008823:Micelles; D010084:Oxidation-Reduction; D011092:Polyethylene Glycols; D011198:Solanum tuberosum / Q000201:enzymology; D013025:Glycine max / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D003902:Detergents; D008041:Linoleic Acids; D008823:Micelles; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; D019787:Linoleic Acid; D008084:Lipoxygenase