Reaction products of the two LOX enzymes and mutants with [1-14C]linoleic acid or [1-14C]arachidonic acid were analyzed on an Agilent 1100 HPLC equipped with a diode array detector connected online to a Radiomatic FLO-ONE A-100 radioactive detector. Straight-phase HPLC analysis used a Beckman Ultrasphere silica column (25 × 0.46 cm) eluted at a flow rate of 1 ml/min with hexane/isopropanol/acetic acid (100/2/0.1, by volume) with UV detection at 205, 220, 235 and 270 nm. Reversed-phase HPLC used a Waters Symmetry C18 5-μm column eluted at 1 ml/min with a solvent of methanol/water/acetic acid (80/20/0.01, by volume), and finally with methanol to elute unreacted substrate. Chiral analysis was performed on HODE methyl esters using a Daicel Chiralpak AD column (25 × 0.46 cm) eluted with hexane/methanol (100/2, by volume) at a flow rate of 1 ml/min, with UV detection at 235 nm [15 (link)].
Protocol detail
HPLC Analysis of LOX Enzyme Reaction Products
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Acetic acid
Arachidonic acid
Chiralpak ad
Enzymes
Esters
Hexane
Hplc
Isopropanol
Linoleic acid
Methanol
Radioactive
Silica
Solvent
Corresponding Organization : Vanderbilt University Medical Center
Other organizations : Michigan State University, Keio University
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Variable analysis
independent variables
- Reaction products of the two LOX enzymes and mutants
- Reaction products analyzed on an Agilent 1100 HPLC equipped with a diode array detector connected online to a Radiomatic FLO-ONE A-100 radioactive detector
- Straight-phase HPLC analysis used a Beckman Ultrasphere silica column (25 × 0.46 cm) eluted at a flow rate of 1 ml/min with hexane/isopropanol/acetic acid (100/2/0.1, by volume) with UV detection at 205, 220, 235 and 270 nm
- Reversed-phase HPLC used a Waters Symmetry C18 5-μm column eluted at 1 ml/min with a solvent of methanol/water/acetic acid (80/20/0.01, by volume), and finally with methanol to elute unreacted substrate
- Chiral analysis was performed on HODE methyl esters using a Daicel Chiralpak AD column (25 × 0.46 cm) eluted with hexane/methanol (100/2, by volume) at a flow rate of 1 ml/min, with UV detection at 235 nm
- No positive or negative controls were explicitly mentioned in the provided information.
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