Determination of the activity of the cytochrome p450 enzyme geraniol 10-hydroxylase in plants by high-performance liquid chromatography

✍ Scribed by Graziella Collu; Hugo H. J. Bink; Paulo R. H. Moreno; Robert van der Heijden; Robert Verpoorte

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 80 KB
• Volume: 10
• Category: Article
• ISSN: 0958-0344
• DOI: 10.1002/(sici)1099-1565(199911/12)10:6<314::aid-pca475>3.0.co;2-z

✦ Synopsis

The cytochrome P450 enzyme geraniol 10-hydroxylase plays an important role in the biosynthesis of pharmaceutically important alkaloids in Catharanthus roseus. An HPLC assay was developed for this enzyme based on the UV detection of the product 10-hydroxygeraniol after its separation from the substrate geraniol on a reversed-phase C-18 column. Furthermore, this system can be used for the UV detection of nerol, which is also a substrate for geraniol 10-hydroxylase, and its product 10-hydroxynerol. The presence of interfering enzymes which epoxidize rather than hydroxylate geraniol and nerol could also be detected. The developed HPLC assay was validated with respect to the incubation time (linear up to 45 min) and the amount of protein added per incubation (linear up to 400 mg of protein). The HPLC assay will be a useful tool during the purification of geraniol 10-hydroxylase from cell suspension cultures of Catharanthus roseus.