The Synthesis of a 5′-Deoxyadenosylcobalamin-agarose Adsorbent and Its Utility in the Purification of Ribonucleotide Reductase

A 5'-deoxyadenosylcobalamin-agarose adsorbent has been prepared for the purification by affinity chromatography of enzymes which require 5'-deoxyadenosylcobalamin as a coenzyme. The synthesis involved the covalent attachment of cyanocobalamin to agarose through a 12 carbon chain. This hydrocarbon chain was attached to the corrin nucleus by reacting diaminododecane with cyanocobalamin e-carboxylic acid in the presence of water-soluble carbodiimide. The substituted cobalamin was then coupled to agarose activated by cyanogen bromide. The complex was reduced with chromous chloride and reacted with 5'-O-p-tolylsulfonyl-adenosine to yield the desired adsorbent. This adsorbent has been successfully used in the purification of ribonucleotide reductase from Lactobacillus leichmannii, which requires 5'-deoxyadenosylcobalamin as a coenzyme. Ribonucleotide reductase is retained by the adsorbent if it is applied in the presence of a dithiol and an allosteric effector. The enzyme is readily eluted with a solution containing only buffer and dithiol.