Conditions Promoting Metal‐Catalyzed Oxidations during Immobilized Cu—Iminodiacetic Acid Metal Affinity Chromatography

Abstract

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

Lewis Acids as Catalysts in Oxidation Reactions: From Homogeneous to Heterogeneous Systems

“…634 Magnesium can act as Lewis acid and through binding with a carbonyl group can control the stereochemistry of the oxidation of NADH analogues by p-benzoquinones. 635 The axis of the carbonyl group bound to the dihydropyridine heterocycle forms a dihedral angle of 55°.…”

Evaluation of IDA-PEVA hollow fiber membrane metal ion affinity chromatography for purification of a histidine-tagged human proinsulin

“…has been reported by Krishnamurthy et al [30] and Bush and Lumpkin [31]. The addition of these reducing agents is relevant to bioprocess as thiol reducing agents such as dithiothreitol, glutatione, sulfite, and ␤-mercaptoethanol in the millimolar range are often used for refolding and solubilization of inclusion bodies [30]. The results obtained by Krishnamurthy et al [30] imply that Cu 2+ -IDA chromatography may result in a damaged protein product when using crude extract containing reductants and/or oxidants.…”

“…The occurrence of MCO reactions in IMAC systems for immobilized Cu 2+ -IDA in the presence of reducing agents such as ascorbate and thiol agents (GSH, cysteine, etc.) has been reported by Krishnamurthy et al [30] and Bush and Lumpkin [31]. The addition of these reducing agents is relevant to bioprocess as thiol reducing agents such as dithiothreitol, glutatione, sulfite, and ␤-mercaptoethanol in the millimolar range are often used for refolding and solubilization of inclusion bodies [30].…”

“…The addition of these reducing agents is relevant to bioprocess as thiol reducing agents such as dithiothreitol, glutatione, sulfite, and ␤-mercaptoethanol in the millimolar range are often used for refolding and solubilization of inclusion bodies [30]. The results obtained by Krishnamurthy et al [30] imply that Cu 2+ -IDA chromatography may result in a damaged protein product when using crude extract containing reductants and/or oxidants. In the case of proteins, MCO reactions occur at metal binding sites on the protein, and the oxidative product attacks the side chains of amino acids at that site [32,33].…”

Inactivation of herpes simplex type 1 gene vector on immobilized metal affinity chromatography: Oxidative damage by hydroxyl free radicals and its prevention

“…Thus, there are possibilities that the metal ions could trigger free radical generation and cause damage to the biological product which is being purified by IMAC. Inactivation of lactate dehydrogenase has been reported when exogenous hydrogen peroxide or reducing agents such as ascorbate, glutathione, or cysteine were intentionally introduced into IDA-Cu 2þ column to create the MCO conditions (Bush and Lumpkin, 1998;Krishnamurthy et al, 1995). However, no inactivation of lactate dehydrogenase was observed during the actual IMAC purification in which no exogenous reducing agent was deliberately introduced (Bush and Lumpkin, 1998;Krishnamurthy et al, 1995).…”