CONDITIONS PROMOTING METAL-CATALYZED OXIDATIONS DURING IMMOBILIZED CU-IMINODIACETIC ACID METAL AFFINITY-CHROMATOGRAPHY

Many solutions that contain oxygen and/or hydrogen peroxide, transitio n metals, and reductants undergo metal-catalyzed oxidation (MCO) react ions. These reactions produce highly reactive radical intermediates wh ich can cause damage to a variety of biomolecules. Some of the types o f damage caused by MCO reactions to proteins are activity losses, irre versible amino acid modifications, increased susceptibility to proteol ysis, and/or fragmentation. The occurrence of such reactions in immobi lized metal affinity chromatography (IMAC) systems has not been report ed, nor has it been well studied. We report here enzyme activity studi es of lactate dehydrogenase (LDH) during chromatography on an immobili zed Cu2+-iminodiacetic acid (IDA) metal affinity column and document t he occurrence of MCO reactions under various chromatography conditions . Chromatography in the presence of the reducing agent ascorbate or th e oxidant hydrogen peroxide caused LDH inactivation, and the presence of both reagents greatly enhanced the loss of activity. Increasing con centrations of reducing agent or hydrogen peroxide led to increased le vels of damage. Chromatography under anaerobic conditions reduced LDH inactivation. Enzyme inactivation on the column was consistent with ac tivity losses observed in solutions containing dissolved Cu2+-IDA. Oth er reducing agents such as glutathione, beta-mercaptoethanol, and cyst eine also caused LDH inactivation during chromatography. During chroma tography in the presence of a reducing agent and/or peroxide, Cu+ and hydroxyl radicals were generated on the column and metal ions were rem oved from the column. Studies with the Cu+-specific chelator bicinchon inic acid indicated that Cu+ was an essential component for the observ ed protein inactivation. The loss of enzyme activity in the presence o f ascorbate and/or peroxide is most likely due to the occurrence of MC O reactions on the column. During chromatography in the absence of add ed reagents, the loss. of LDH activity and the occurrence of MCO react ions were not detected over the chromatography times used in this stud y. However, LDH inactivation did occur in solutions containing dissolv ed Cu2+-IDA. An understanding of the conditions under which MCO reacti ons occur during IMAC would aid the design of better downstream proces sing operations utilizing metal affinity methods.