Many solutions that contain oxygen and/or hydrogen peroxide, transition metals, and reductants undergo metal-catalyzed oxidation (MCO) reactions. These reactions produce highly reactive radical intermediates which can cause damage to a variety of biomolecules. Some of the types of damage caused by MCO reactions to proteins are activity losses, irreversible amino acid modifications, increased susceptibility to proteolysis, and/or fragmentation. The occurrence of such reactions in immobilized metal affinity chromatography (IMAC) systems has not been reported, nor has it been well studied. We report here enzyme activity studies of lactate dehydrogenase (LDH) during chromatography on an immobilized Cu2+-iminodiacetic acid (IDA) metal affinity column and document the occurrence of MCO reactions under various chromatography conditions. Chromatography in the presence of the reducing agent ascorbate or the oxidant hydrogen peroxide caused LDH inactivation, and the presence of both reagents greatly enhanced the loss of activity. Increasing concentrations of reducing agent or hydrogen peroxide led to increased levels of damage. Chromatography under anaerobic conditions reduced LDH inactivation. Enzyme inactivation on the column was consistent with activity losses observed in solutions containing dissolved Cu2+-IDA. Other reducing agents such as glutathione, P-mercaptoethanol, and cysteine also caused LDH inactivation during chromatography. During chromatography in the presence of a reducing agent and/or peroxide, Cu+ and hydroxyl radicals were generated on the column and metal ions were removed from the column. Studies with the Cu+-specific chelator bicinchoninic acid indicated that Cu+ was a n essential component for the observed protein inactivation. The loss of enzyme activity in the presence of ascorbate and/or peroxide is most likely due to the occurrence of MCO reactions on the column. During chromatography in the absence of added reagents, the loss of LDH activity and the occurrence of MCO reactions were not detected over the chromatography times used in this study. However, LDH inactivation did occur in solutions containing dissolved Cu2+-IDA. An understanding of the conditions under which MCO reactions occur during IMAC would aid the design of better downstream processing operations utilizing metal affinity methods.8756-7938/95/3011-0643$09.00/0 0

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.