Preparation and characterisation of P450 3A4
This study demonstrates that highly pure P450 3A4 can be prepared utilising either of the detergents Emulgen 911 (E911) or CHAPS as the solubilising agent, and that CHAPS, a steroidal detergent, binds to P450 3A4 but is less inhibitory than E911, most likely because of its low affinity. In addition this work highlights, for the first time, that CHAPS-solubilised P450 3A4 is not a homogeneous preparation, and is in fact a mixture of oligomers, the size of the oligomeric state being affected by the amount of detergent used to purify the enzyme. Despite this aggregation, however, the preparations of P450 3A4 were shown to be active with respect to binding carbon monoxide in the reduced for, oxidising a profluorescent substrate (7-BQ), and producing an optical spin state change upon substrate binding. Optical binding studies have suggested a correlation between a compound's affinity for P450 3A4 and its solubility, i.e. the more soluble the substrate the weaker it will bind to this protein, as has been suggested in the past. These studies have also indicated that, (i) the affinity of a substrate may be enhanced by the presence of a nitrogen that can coordinate to the iron, (ii) the active site is unlikely to be merely a large cavity where substrates can bind, (iii) the cooperative behaviour of testosterone and amitriptyline with P450 3A4 is dependent upon the buffer conditions used and (iv) one or more substrate molecules may be interacting with this protein at once, and at distinct binding sites, although the nature of these sites cannot be determined. In addition, NMR relaxation studies were carried out in an attempt to determine haem iron to substrate proton distances for P450 3A4. However, it was not possible to achieve this because the systems under investigation were all in slow/intermediate exchange on the NMR time scale, possibly because the protein samples were aggregated.