Abstract:
Flavokinase (EC 2.7.1.26) converts riboflavin to FMN and FAD synthetase (EC 2.7.7.2) converts FMN to FAD. The presence of these enzymes was determined in the human term placenta. Placentas were obtained after vaginal or caesarean births. The placental tissue was scraped from the maternal side and the tissue was homogenised and centrifuged at 18,000 g. The 35-80% ammonium sulphate fraction, obtained from the supernatant, contained l00% of the flavokinase activity and possibly contained FAD synthetase activity. The presence of FMN and FAD as products of these assays was indicated by thin layer chromatography, high performance liquid chromatography and fast atom bombardment mass spectrometry.
The conditions for the assay of placental flavokinase were examined in incubates with radiolabelled riboflavin as substrate and separation of product FMN by thin layer chromatography Placental flavokinase had an alkaline pH optimum but was routinely assayed at less than the optimum pH to minimise the effects of alkaline phosphatase. FMN production in the assay reached a maximum after l0 min incubation. ATP was essential for the activity of the enzyme.
Placental flavokinase was partially purified using affinity chromatography. The undialysed 35-80% ammonium sulphate precipitate was mixed with blue Sepharose matrix and the protein was eluted from the column with water. The active fractions were combined and applied to an agarose C-8 ATP column. Application of a combination of these chromatography techniques gave an approximately 350-fold purification of the flavokinase from the placental homogenate. Gel permeation chromatography on a Biogel P-60 column indicated a MW of approximately 29,000 for the partially purified flavokinase. SDS polyacrylamide gel electrophoresis of the purified fractions also indicated the presence of protein bands at this MW range.
In summary, the work presented in this thesis provides evidence that the human term placenta synthesises FMN and FAD from riboflavin and describes the purification and some of the properties of the flavokinase that catalyses the first step in the synthesis of the two coenzymes.
