Assay Conditions for Bufuralol 1'-Hydroxylation

Bufuralol 1’-hydroxylation is a low K_m, high turnover assay for human CYP2D6. The apparent K_m for the CYP2D6-catalyzed reaction is 5-10  uM. The turnover number (per unit cDNA-expressed CYP2D6 is 10 to 30 min^-1). When performing kinetic analyses near the apparent K_m, protein concentration and incubation time should be chosen to avoid excessive metabolism of substrate. GENTEST supplies 1’-hydroxybufuralol metabolite standard for quantitation of the assay results.

Solutions
I.     1 mM Bufuralol in 0.1 M potassium phosphate pH 7.4
II.    20 mg/ml Glucose 6-phosphate, 20 mg/ml NADP, 13.3 mg/ml MgCl2-H2O
III.   40 U/ml Glucose 6-phosphate dehydrogenase in 5 mM sodium citrate (tribasic)
IV.   0.1 M Potassium phosphate pH 7.4
V.    70% Perchloric acid

Incubation Conditions (for 1 ml Final Volume)
50 ul          Solution II.
10 ul          Solution III.
100 ul        Solution I. (Final concentration 100 uM, a saturating concentration)
xx ul          Enzyme. (human liver microsomes or cDNA-expressed)
840 - xx ul  Solution IV.

Mix and prewarm to 37^oC all solutions except enzyme. Initiate incubation with the addition of enzyme. After the desired incubation time, stop the reaction by the addition of 100 ul of solution V and cool on ice. Centrifuge 12000 x g for 4 minutes to precipitate protein. Analyze the supernatant for product formation by HPLC separation with fluorescence detection. Recommended range of injection volumes - 10 to 150 ul.

HPLC Conditions
Mobile Phase A: 30% Acetonitrile, 70% water, 1 mM Perchloric acid (See Note 1)
Isocratic
Column: Nucleosil C18, 4.6 x 250 mm, 5 um particle size (see Note 2)
Temperature: 50^oC (see Note 3)
Flow Rate: 1 ml/min
Detector: Fluorescence: Excitation at 252 nm and Emission at 302 nm
Retention Times: 1’-Hydroxybufuralol, 6 minutes; Bufuralol, 20 minutes

Note 1
Bufuralol and 1’-hydroxybufuralol are being chromatographed as ion pairs with perchlorate as the counter ion.
Note 2
1’-Hydroxybufuralol and bufuralol are easily separated and most C18 columns should be adequate for the purpose. However, some adjustment in mobile phase acetonitrile concentration may be desired.
Note 3
Column temperature can range from room temperature to 50^oC. The use of a controlled, elevated temperature provides greater reproducibility in retention times and lower column back pressures.

Reference
Anal. Biochem. (1987) 162, 24