A simplified squalene epoxidase assay based on an HPLC separation and time-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 determination 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 to provide an ideal mobile phase, into which aqueous enzyme reaction mixture aliquots could be injected. Squalene, the natural substrate for squalene epoxidase, may be quantitatively determined within the concentration range 0-30 microM, with a calibration curve exhibiting an r2 (where r2 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 enzyme assay reaction mixture, so facilitating its identification and quantification. In this way HPLC was used to follow enzymic squalene consumption within aliquots taken over a 30-min period. Previously reported squalene epoxidase assays rely on the radiolabeling and subsequent monitoring of squalene as it is metabolized by the enzyme. A highly simplified enzyme assay for squalene epoxidase is therefore reported.
Grieveson
L A
LA
Manchester Materials Science Centre, University of Manchester Institute of Science and Technology, United Kingdom.
Ono
T
T
Sakakibara
J
J
Derrick
J P
JP
Dickinson
J M
JM
McMahon
A
A
Higson
S P
SP
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002855:Chromatography, Thin Layer / Q000379:methods; D010105:Oxygenases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013185:Squalene / Q000031:analogs & derivatives / Q000032:analysis* / Q000378:metabolism; D050603:Squalene Monooxygenase; D013997:Time Factors
C002821:2,3-oxidosqualene; D013185:Squalene; D010105:Oxygenases; D050603:Squalene Monooxygenase