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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

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