Preparation of substrate stock solutions and incubation conditions with individual HLM
Substrates | Weight/Volume | Organic Solvent | Stock | Incubationa | Incubation Time | Reaction Quench Solutionb |
|---|---|---|---|---|---|---|
| mg/ml | mM | μM | min | |||
| APAP | 151/1 | Methanol | 1000 (500×) | 2000 | 45 | 1 volume A |
| AZT | 33.38/0.5 | Methanol | 250 (500×) | 500 | 45 | 1 volume A |
| Bilirubin | 8.4/1.44 | DMSO | 10 (1000×) | 10 | 45 | 2 volume B |
| Estradiol | 11.5/1.415 | Methanol | 30 (1000×) | 30 | 15 | 2 volume A |
| Ethinyl estradiol | 4.45/1.0 | Methanol | 15 (1000×) | 15 | 45 | 1 volume A |
| Lorazepan | 5.6/0.174 | Methanol | 100 (4000×) | 25 | 15 | 1 volume A |
| Muraglitazar | 5.4/1.046 | Methanol | 10 (1000×) | 10 | 42 | 2 volume A |
| SN-38 | 5.3/0.45 | DMSO | 30 (1000×) | 30 | 15 | 1 volume C |
DMSO, dimethyl sulfoxide.
↵ a Incubation concentrations were based on literature-reported Km values as described under Material and Methods.
↵ b Quenching solutions were: A, 3% acetic acid in acetonitrile; B, 6% acetic acid in ethanol; C, 1% acetic acid in acetonitrile containing D3-labeled testosterone.