| Assay Method Information | |
| | Optical Titration Assay |
| Description: | Optical titrations to determine the binding affinity (KD values) of ligands were carried out on a Cary 60 UV−visible spectrophotometer (Varian, UK) according to a previously described procedure [McLean et al., J. Inorg. Biochem., 91:527-541]. Ligands were prepared as stock solutions typically 0.1−100 mM) in DMSO and added as 0.2 μL aliquots to cuvettes containing either a solution of CYP144A1 (∼4-8 μM) in 100 mM KPi (pH 7.0), containing 10 mM KCl, or to buffer alone. DMSO concentrations did not exceed 1% v/v of the final titration volume (1 mL), and the absorbance spectrum of CYP144A1 was not affected within this range. Spectra were recorded continuously between 800 and 250 nm at 25 °C. |
| Affinity data for this assay | |
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