Purification of the flavoproteins 6-hydroxy-D- and 6-hydroxy-L-nicotine oxidase using hydrophobic affinity chromatography.

A systematic study of the homologous series of omega-aminoalkyl-agaroses revealed differences in the affinities of 6-hydroxy-D- and 6-hydroxy-L-nicotine oxidase. In contrast to supports with nonpolar alkyl chains, omega-aminoalkyl-agarose showed high affinity towards the L-specific enzyme, while the D-specific oxidase was bound most firmly by omega-aminododecyl-agarose. 6-Hydroxy-L-nicotine oxidase could be desorbed by 1.3M NaCl only in the presence of the substrate L-6-hydroxynicotine. Using the omega-aminoalkyl-agarose, a complete separation of the enantiozymes was accomplished and an efficient purification procedure for both oxidases established.