Abstract
Electron-transferring flavoprotein purified from the anaerobic bacterium Megasphaera elsdenii contains one molecule of coenzyme FAD and can bind additional one molecule of FAD to become the holoprotein. The additionally bound FAD exhibits an anomalous absorption spectrum which shows a main absorption band at 400 nm with a shoulder at 450 nm. The flavin released from holoprotein by potassium bromide or guanidine hydrochloride was identified to be normal FAD judging from its absorption spectrum and the retention time on reverse phase chromatography. This result indicates that the unusual spectrum is not due to a chemical modification of the flavin ring but the flavin environment in the protein. The apoprotein was prepared from the holoprotein by cycles of ultrafiltration and dilution with guanidine hydrochloride solution. Dilution of the guanidine-denatured apoprotein with a buffer solution containing FAD resulted in the holoprotein which showed the unusual absorption spectrum like the original holoprotein. This result excludes the possibility that unknown small molecules contained in the holoprotein affect the flavin absorption spectrum. Mammalian electron-transferring flavoprotein contains one FAD and one AMP, unlike M. elsdenii protein. AMP-free mammalian protein, prepared by denaturation and renaturation, showed under acidic conditions an absorption spectrum similar to the unusual spectrum of M. elsdenii protein. This observation suggests a common flavin environment between mammalian and M. elsdenii proteins. [Jpn J Physiol 54 Suppl:S65 (2004)]
