Structural Damage to Lactate Dehydrogenase during Copper Iminodiacetic Acid Metal Affinity Chromatography

The stability of the enzyme lactate dehydrogenase (LDH) was evaluated by measuring structural damage and activity loss after exposure to copper−iminodiacetic acid (IDA) immobilized metal affinity chromatography (IMAC) under oxidizing conditions at pH 7.0. Oxidizing conditions were produced by adding reductants commonly employed in bioprocessing and biomedical applications (glutathione, β‐mercaptoethanol, dithiothreitol, cysteine, or ascorbate) and/or hydrogen peroxide to the mobile phase. Most of these additives have been shown recently to give rise to metal‐catalyzed oxidation (MCO) reactions on copper−iminodicaetic acid IMAC columns. Structural damage in the form of increased susceptibility to proteolytic degradation, fragmentation, and cross‐linking were measured. Increased sensitivity to proteolysis was significant in virtually all cases tested, even when activity remained high (>95% specific activity recovered). In contrast fragmentation and cross‐linking were minimal in all cases, even when activity was low (