Reversed phase hplc system

The sterol 14α-demethylase activity of M. capsulatus CYP51fx was reconstituted with radiolabeled (3-³H) lanosterol, eburicol, and obtusifoliol, specific activity ∼4,000 dpm nmol⁻¹, as described previously for CYP51 from My. tuberculosis (Lepesheva et al. 2001 (link)), except that the sterols were added from a 0.5-mM solution in 45% (w/v) HPCD. Escherichia coli flavodoxin and flavodoxin reductase (molar excess over P450 18 and 2, respectively), served as electron donor partners. The enzyme/substrate molar ratio was 1/25 (2/50 µM). The final reaction volume was 500 µl and contained 20 mM MOPS (pH 7.4), 50 mM KCl, 5 mM MgCl₂, 10% (v/v) glycerol, 0.4 mg ml⁻¹ isocitrate dehydrogenase, and 25 mM sodium isocitrate. The reaction was initiated by addition of 5 mM NADPH and stopped by extraction of the sterols with ethyl acetate. The extracted sterols were analyzed by a reversed-phase HPLC system (Waters) equipped with a β-RAM detector (INUS Systems). In order to verify the ability of the fusion M. capsulatus ferredoxin domain to transfer electrons to the P450 domain, we used spinach ferredoxin reductase (Jackson et al. 2002 (link)). In this case, the CYP51fx concentration was increased to 10 µM, the concentration of ferredoxin reductase was 25 µM. At these conditions, 25% of lanosterol conversion was observed in a 1-h reaction.