PURIFICATION AND CHARACTERIZATION OF A RIBOFLAVIN-BINDING PROTEIN FROM FLAGELLA OF EUGLENA-GRACILIS

Phototaxis of the flagellate Euglena gracilis has been thought to be mediated by flavin photoreceptor molecules localized in the paraflagellar body (PFB). From isolated flagella of Euglena a riboflavin (RF)-binding protein was solubilized and purified using nonionic detergents, high ionic strength, affinity chromatography and standard column separations. Sodium dodecyl sulfate gel electrophoresis showed an apparent molecular weight of 68 kDa for the binding protein. Its hydrophobicity was confirmed by Triton X-114 phase partitioning. Binding affinity for tritiated RF was high in the oxidized state (K-D = 4 nM) as well as under reducing conditions in the presence of dithionite (K-D = 6 nM). Affinities towards flavin mononucleotide and flavin adenine dinucleotide were lower. Based on binding data and on estimates of the purified 68 kDa polypeptide, approximately 10(6) flavin-binding sites were determined per one flagellum. Evidence is discussed that the flavin-binding protein is part of the entire flagellar membrane and does not reside in the PFB. If not the photoreceptor, the flagellar RF-binding protein may have a functional role in the biochemical chain leading from the reception of the phototactic stimulus to the motile response.