Loss of scup liver spectral P-450 stimulated by TCB and NADPH
| Additions to Reaction Mixtures | P-450 | P-420 Concentration (range) | Heme | ||
|---|---|---|---|---|---|
| Concentration | Loss (range) | Concentration | Loss (range) | ||
| nmol/mg | % | nmol/mg | nmol/mg | % | |
| Acetone | 0.89 ± 0.33 | ND1-a | 1.44 ± 0.52 | ||
| NADPH | 0.73 ± 0.35 | 22 ± 9 | 0.04 ± 0.07 | 1.26 ± 0.49 | 16 ± 12 |
| (17–47) | (0.00–0.19) | (0–37) | |||
| TCB | 0.91 ± 0.46 | ND | 1.34 ± 0.46 | ||
| TCB + NADPH | 0.48 ± 0.221-150 | 49 ± 7 | 0.07 ± 0.10 | 1.05 ± 0.40 | 28 ± 16 |
| (32–63) | (0.00–0.29) | (1–48) | |||
To determine whether there was a loss of P-450 or heme, microsomes were incubated at 0.4 mg/ml in buffer (50 mM Tris and 0.1 mM EDTA, pH 7.6) at a final volume of 1 ml, at 30°C. Acetone (0.5%), TCB (17 μM), and NADPH (0.7 mM) were added in various combinations. Reactions were allowed to proceed for 30 min, at which time the rate of loss of P-450 become nonlinear. Total P-450 concentration was determined from dithionite-difference spectra. Heme was determined in reaction mixtures by the pyridine hemochrome assay. P-450/P-420 determinations were made on nine pools of microsomes, and initial concentrations ranged from 0.53 to 1.40 nmol/mg. Heme determinations were made on six pools of microsomes, and initial concentrations ranged from 0.60 to 2.04 nmol/mg. Data are means ± S.D.