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, beta-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 (< 50%). As the damage was believed to have been caused primarily by MCO reactions, preventative measures consistent with this reaction pathway were tested. The most successful measure for all of the conditions studied was addition of the Cu+ chelating agent bicinchoninic acid (BCA) to the mobile phase. Decreased contact time with the column decreased damage in the case where glutathione was added. Removal of dissolved oxygen by nitrogen sparging and use of Tris-acetate buffer in place of phosphate had no measurable effect. The success of BCA addition in reducing structural damage and activity loss strengthens the conclusion that MCO reactions can occur on copper-iminodiacetic acid IMAC columns. However, the addition of BCA and the other protective measures described were not successful in eliminating the increased proteolytic susceptibility observed when LDH in buffer was exposed to the copper-charged column with no oxidizing additives. This suggests that at least one other pathway for damage exists. This damage is difficult to detect as it did not cause statistically significant losses in enzymatic activity, fragmentation, or cross-linking.