Quantification of the production of dihydrokaempferol by flavanone 3-hydroxytransferase using capillary electrophoresis.
A sensitive method using capillary electrophoresis for the separation, detection, and quantification of dihydrokaempferol (1) is reported. Well-resolved, sharp symmetrical peaks were obtained in grapefruit leaf extracts for 1, naringenin (2), and the internal standard, naringin (3). Long columns were required to resolve 1 from 2 in crude enzyme reactions and this resulted in run times of 60 min. The limit of detection for 1 was found to be 1.44 ng/microL (4.2 pg). The method showed excellent linearity and reproducibility. The method was used to determine the activity of flavanone 3-hydroxytransferase (F3H) in leaf tissue of grapefruit by quantification of the production of dihydrokaempferol in controlled time course reactions. The sensitivity of the method makes it adaptable to assaying F3H activity in individual young seedlings and/or in small tissue samples and requires only 100 mg of tissue.
Owens
Daniel K
DK
Department of Biological Sciences, East Tennessee State University, Johnson City, TN 37614, USA.
Hale
Tracy
T
Wilson
Lori J
LJ
McIntosh
Cecilia A
CA
D002957:Citrus / Q000737:chemistry / Q000201:enzymology; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D044950:Flavanones; D005419:Flavonoids / Q000032:analysis / Q000378:metabolism*; D015394:Molecular Structure; D018515:Plant Leaves / Q000737:chemistry / Q000201:enzymology; D011794:Quercetin / Q000031:analogs & derivatives* / Q000032:analysis* / Q000378:metabolism*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012995:Solubility; D013696:Temperature; D013997:Time Factors
D044950:Flavanones; D005419:Flavonoids; C080220:aromadedrin; D011794:Quercetin; C005273:naringenin