Abstract

For the demonstration of a novel riboflavin kinase assay method based on the bacterial bioluminescence, partially purified riboflavin kinase was prepared from bovine liver through ammonium sulfate precipitation and DEAE-cellulose ion exchange chromatography. Using bacterial luciferase from Photobacterium phosphoreum and the dithionite reduction method, an easy, safe, and fast assay method was established. The optimal temperature, pH, Km values for riboflavin and ATP of bovine liver riboflavin kinase determined with this luminescence method were 35 C, pH 7, 15.3 uM and 8.3 uM, respectively. The detection limit of FMN produced by riboflavin kinase was in the range of 200 pM to 4 uM which is comparable to the HPLC-fluorescence detection method, while the detection time for each assay was less than 15 sec compared to the HPLC method which requires at least 10 min for completion.

• Keywords: Riboflavin kinase; bioluminescence