Purification and detection of vitamin B 12 analogs.

Cobamides are a family of enzyme cofactors that are required by organisms in all domains of life. Over a dozen cobamides exist in nature although only cobalamin (vitamin B), the cobamide required by humans, has been studied extensively. Cobamides are exclusively produced by a subset of prokaryotes. Importantly, the bacteria and archaea that synthesize cobamides de novo typically produce a single type of cobamide, and furthermore, organisms that use cobamides are selective for certain cobamides. Therefore, a detailed understanding of the cobamide-dependent metabolism of an organism or microbial community of interest requires experiments performed with a variety of cobamides. A notable challenge is that cobalamin is the only cobamide that is commercially available at present. In this chapter, we describe methods to extract, purify, and quantify various cobamides from bacteria for use in laboratory experiments.