Immobilized metal ion affinity chromatography on Co2+-carboxymethylaspartate-agarose Superflow, as demonstrated by one-step purification of lactate dehydrogenase from chicken breast muscle

A rapid method for the purification of lactate dehydrogenase from whole chi cken muscle extract in one chromatographic step is reported. The purificati on procedure can be accomplished in less than I h. A new type of immobilize d metal ion affinity chromatography adsorbent is used that can be utilized at linear flow rates higher than 5 cm/min, The final preparation of the enz yme was with purity higher than 95 % as ascertained by SDS-PAGE, Three immo bilized metal ions (Ni2+, Zn2+ and Co2+) were compared for their binding pr operties towards the purified enzyme. The binding site of the enzyme for im mobilized intermediate metal ions was determined after cleavage with CNBr a nd binding studies of the derivative peptides on immobilized Co2+. A peptid e located on the hi-terminus of the enzyme, implicated in the binding, has great potential as a purification tag in fusion proteins.