13.Assay of LTB4 12-hydroxy-dehydrogenase（横溝岳彦）
@Stock solutions
500 mM Potassium phosphate buffer pH7.5
10m M NADP in 0.1 mM Tris-HCl pH 7.5
1.5 mM LTB4 in ethanol
Stopper 4 μM 13-OH-linoleic acid in Ethyl acetate
Solvent Y CH3CN:H2O:Acetic acid=50:50:0.01, 0.01% EDTA-Na2
(w/v), pH adjusted to 5.6 by 1 N Ammonium water
@Reaction mixture (Total 100 μl) contains;
100 mM Potassium phosphate buffer pH7.5 (20 μl of stock)
1 mM NADP in 0.1 mM Tris-HCl pH 7.5 (10 μl of stock)
Enzyme solution
Water
Initiate reaction by adding 3 nmol (2 μl) of LTB4 solution.
Incubate at 37 ｡C for various time
Stop the reaction by adding 200 μl of stopper
Centrifuge at 12,000 rpm for 5 min
Take 150 μl of the supernatant and evaporate by the concentrator
Add 200 μl of Solvent Y, vortex and inject 100 μl to HPLC
HPLC condition;
Flow 1.0 ml/min
Monitor 320 nm and 235 nm
Column Cosmosil 5C18-AR, 4.6 x 150 mm
Column temp. 37 ｡C
Approximate retention time ; Wavelength
LTB4 5.8 min (235 nm)
12-Oxo-LTB4 7.2 min 316 nm
Standard 18.0 min 235 nm
Calculation
320 nm product; 0.508xArea(oxo)/Area(St)
235 nm products; 0.683xArea(dihydro)/Area(St)
(Ref.)
1. Yokomizo, et. al, J. Biol. Chem. (1993) 268, p18128-18135
2. Yokomizo, et. al, J. Biol. Chem. (1996) 271, p2844-2850