Binding spectra characteristics of CYP3A4 and CYP2C9 with type II ligands
P450 + Liganda | Absolute Spectra | Difference Spectra | Dissociation Constant KS | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Soret λmax | % Change in Soret | ΔA424-390b | Peak λmax | Trough λmax | ΔAmaxc | Difference Spectra | Absolute Spectra | Equation Used for Fitting and Hill Coefficient | |||||||
| nm | ∈ | nm | μM | ||||||||||||
| CYP3A4 a | 417 | 0 | |||||||||||||
| Imidazole (25 mM) | 424 | -1.9 | Positive | 434 | 409 | 0.0282 | 572 ± 77 | N.D.d | 1 | ||||||
| Aniline (25 mM) | 422 | -5.2 | Positive | 429 | 410 | 0.0214 | 1701 ± 217 | N.D.d | 1 | ||||||
| 1,2,4-Triazole (40 mM) | 422 | -4.9 | Positive | N.D.e | 410 | 0.0116 | 65.1 ± 13.4 | 49.3 ± 14.3 | 2,2 | ||||||
| 0.0025 | |||||||||||||||
| PH-302 (0.05 mM) | 424 | -7.6 | Positive | 431 | 409 | 0.0198 | 5.5 ± 0.3 | 6.6 ± 0.3 | 1 | ||||||
| Itraconazole (0.003 mM) | 421 | -1.4 | Positive | 426 | 407 | 0.0130 | 0.456 ± 0.008 | 0.645 ± 0.014 | 5 (n = 1.8) | ||||||
| Fluconazole (0.1 mM) | 420 | -0.5 | Positive | 426 | 400 | 0.0112 | 9.8 ± 0.5 | 25.2 ± 1.9 | 1 | ||||||
| CYP2C9 a | 417 | 0 | |||||||||||||
| Imidazole (25 mM) | 424 | -17.7 | Positive | 434 | 412 | 0.0290 | 453 ± 50 | 213 ± 83 | 1 | ||||||
| Aniline (25 mM) | 420 | -15.8 | Negative | N.D.e | 412 | 0.0152 | 1757 ± 103 | N.D.d | 1 | ||||||
| 1,2,4-Triazole (40 mM) | 419 | -16.5 | Negative | N.D.e | 412 | 0.0161 | 55.8 ± 22.1 | 39.6 ± 7.3 | 2,1 | ||||||
| 0.0027 | |||||||||||||||
| PH-302 (0.5 mM) | 420 | -26.0 | Negative | N.D.e | 412 | 0.0094 | 31.2 ± 6.0 | 11.6 ± 4.1 | 3 | ||||||
| 0.0033 | |||||||||||||||
| Itraconazole (0.1 mM) | 418 | -28.7 | Negative | 432 | 410 | N.D.d | N.D.d | 15.2 ± 3.1 | 5 (n = 1.7) | ||||||
| Fluconazole (0.5 mM) | 419 | -16.5 | Negative | N.D.e | 412 | 0.0050 | 62.5 ± 12.5 | 7.31 ± 1.78 | 3 | ||||||
| 0.0017 | |||||||||||||||
| Sulfaphenazole (0.1 mM) | 418 | -7.9 | Negative | 426 | 409 | 0.0008 | 0.023 ± 0.017 | 0.238 ± 0.242 | 4 | ||||||
↵ a P450 concentration was 0.5 μM in all experiments and the ligand concentration represents the saturating or maximum concentration used.
↵ b Direction of signal change as the titration progresses.
↵ c ΔAmax is given for saturation of the high-KS/low-affinity site followed by the high-affinity/low-KS site. In some cases, saturation of the high-KS site was not possible without resulting in high DMSO concentrations or precipitation of the enzyme.
↵ d Not determined, or ligand absorbance prevented characterization.
↵ e No distinct peak is observable in difference spectra due to decrease in intensity or lack of red-shift of the low-spin Soret band; baseline was used in calculating ΔAmax and KS.