A SIMPLIFIED SQUALENE EPOXIDASE ASSAY BASED ON AN HPLC SEPARATION ANDTIME-DEPENDENT UV VISIBLE DETERMINATION OF SQUALENE/

A novel and highly simplified enzyme assay for squalene epoxidase (EC 1.14.99.7) has been developed. The assay relies on the UV/visible dete rmination of squalene at 195 nm, as it elutes from an octadecylsilane HPLC column. An acetonitrile/water (95.5/0.5, v/v) mixture was found t o provide an ideal mobile phase, into which aqueous enzyme reaction mi xture aliquots could be injected. Squalene, the natural substrate for squalene epoxidase, may be quantitatively determined within the concen tration range 0-30 mu M, with a calibration curve exhibiting an r(2) ( where r(2) is the square of the Pearson correlation coefficient r) of 0.995. The HPLC retention time for squalene was significantly longer ( >15 min) than that for any other component required to prepare an enzy me assay reaction mixture, so facilitating its identification and quan tification. In this way HPLC was used to follow enzymic squalene consu mption within aliquots taken over a 30-min period. Previously reported squalene epoxidase assays rely on the radiolabeling and subsequent mo nitoring of squalene as it is metabolized by the enzyme. A highly simp lified enzyme assay for squalene epoxidase is therefore reported. (C) 1997 Academic Press.