Purification and analysis of cobamides of Methanobacterium bryantii by high-performance liquid chromatography

Cobamides from Methanobacterium bryantii were purified by isocratic reverse-phase high-performance liquid chromatography at neutral pH in readily extractable buffers. A gradient adaptation of this chromatography system on C-18 muBondapak columns in buffers containing aqueous methanol and 100 mM LiCl readily separated Co alpha-(5-hydroxybenzimidazoyl)-Co beta-cyanocobamide, Co alpha-(5-hydroxybenzimidazoyl)-Co beta-adenosylcobamide, cyanocobalamin, adenosylcobalamin, and methylcobalamin. Aquacobalamin was not resolved from cyanocobalamin by this procedure. The gradient procedure was also useful in following the fate of tritiated cyanocobalamin in cell-free extracts of M. bryantii.