Presentation of: Conditions Promoting Metal Catalyzed Oxidations during Immobilized Cu-Iminodiacetic Acid Metal Affinity Chromatography Authors: Rajesh Krishnamurthy. Rapti D. Madurawe, Kristen D. Bush and Janice Lumpkin Journal: Biotechnology Progress. 1995, 11, 643-650. R. Kennedy

Introduction Affinity chromatography defined Immobilized metal affinity chromatography (IMAC) Proteins that interact with transition metals can be purified via IMAC Cu, Ni, Zn, Co, Fe These metals are immobilized on a chelator IDA, NTA, TED are typical chelators used Amino acid side chains interact with the metal his, lys, cys, pro, arg, met, trp, tyr

Introduction Metal catalyzed oxidation (MCO) reactions can damage proteins. damage includes activity loss, amino acid modification, increased susceptibility to proteolysis, fragmentation. MCO reactions require oxygen, a chelated transition metal and an electron donor. 1st step: oxygen to H2O2, the metal is reduced. 2nd step: peroxide reacts with the reduced metal to form OH radical (Fenton reaction)

Literature Review IMAC shows great utility for protein purification Porath J. et al. Nature. 1975. 258, 598-599. Belew M. and J. Porath. J. Chromatogr. 1990. 516, 333-354. Beitle R. R. and M. M. Ataai. AIChE Symp. Ser. 1992. 88 (290), 34-44. Zawitowska, U. et al. Biotech. Appl. Biochem. 1992. 15, 160-170. Hoculi, E. J. Chromatogr. 1988. 444, 293-302. Immobilized metal + oxygen + reducing agent can cause MCO Dean, R. T. and P. Nicholson. Free Rad. Res. 1994. 20, 83-101. Stadtman, E. R. and B. S. Berlett. J. Biol. Chem. 1991. 266 (26), 17201-17211.

Literature Review MCO reactions in proteins occur at the amino acid side chains of the metal binding sites (lys, cys, pro, arg, met, try, tyr) and can damage the protein. Fucci, L. et al. PNAS. 1983. 80, 1521-1525. Shiner, E. et al. J. Biol. Chem. 1983. 258, 14778-14783. Levine, R. L. et al. PNAS. 1981, 78, 2120-2144.). Amici, A. et al. J. Biol. Chem. 1989. 264 (6), 3341-3346.; Stadman, E. R. Free Rad. Biol. Med. 1990, 9, 315-325.; Schöneich, P. et al. Biochem. Biophys. Acta 1993. 1158, 307-322.; Davies, K. J. et al. J. Biol. Chem. 1987. 262, 9902-9907. Protein damage at these amino acids sites is measurable as activity loss, fragmentation, cross-linking, aggregation, side chain modification (micro-heterogeneity) and increased proteolysis Stadman, E. R. Free Rad. Biol. Med. 1990, 9, 315-325.

Literature Review Porath says: can’t compare IMAC and solutions of chelator-metal-protein and MCO reaction don’t occur on columns (Porath, J. Protein Expression Purif. 1992. 3, 263-281). These authors: MCO reactions should occur on IMAC columns Model system: Lactate Dehydrogenase (LDH) LDH losses activity in solution MCO reactions (Alonso-Llamazares, A. M. et al. Biochem. Int. 1992. 27, 879-889.) LDH is a ubiquitous metabolic protein; there is rapid and reliable activity assay IMAC on Cu-IDA is widely used; Cu is known to participate in MCO reactions, LDH can be purified on Cu_IDA IMAC coloumns Investigation will include multiple reducing agents

Hypothesis An MCO reaction will occur on a Cu-IDA IMAC column. If an MCO reaction occurs on a Cu-IDA IMAC column then LDH will loss activity. If an MCO reaction occurs on a Cu-IDA IMAC column then OH radicals will be seen.

Methodology Chromatography Procedure All buffers and samples treated with Celex100 to remove trace metals. Temperature: Room temperature Buffer A: 0.5 M NaCL, 20 mM Na phosphate, pH 7 Buffer B: Buffer A + 50 mM imidazole, pH 7 Column: HiTrap chelating Cu-IDA, 5 ml Flow rate: 1 ml/min Run time: 5 column volumes Sample volume: 0.2 ml (loop injection) Elution: step gradient (100% B) Sample collection: 3 ml samples collected into EDTA in buffer A on ice (to stop MCO outside column)

Methodology Solution Studies LDH Activity Assay (in duplicate) Final concentrations of 10nM LDH, 0.04 mM CuSO4, 0.4 mM IDA, 0.4 mM BCA, 2 mM ascorbate, and/or 2 mM peroxide in 80 mM Na phosphate pH 7 were incubated at room temperature. At sampling time 0.1 ml aliquots were withdrawn for assay. LDH Activity Assay (in duplicate) All enzyme activity results (%) are compared to a control LDH sample in phosphate buffer (100%) Oxidation of NADH in the presence of pyruvate is measured at 340 nm. LDH activity in units/ml was calculated by (-DA340/min) x (sample volume/reaction volume)(sample dilution)

Methodology Protein Determination Assay (in duplicate) Protein concentration in mg/ml was measured by the micro Lowry method. Standard curve was constructed with BSA.  Specific Activity (calculation) Enzyme specific activity in units/mg was calculated from the ratio of activity to protein concentration. Activity in the results section refers to specific activity compared to the control where the control is 100%.

Methodology Hydrogen Peroxide Assay Hydroxyl Radical Assay Blank was deionized water. Standard curve was constructed with known amounts of MSA in phosphate buffer. OH radicals trapped by DMSO and converted to MSA, MSA is measured at 424 nm. Samples collected from the columns. Mixed with DMSO immediately. Copper Determination Assay Blank was reaction mixtures without copper. Standard curve constructed from known amounts of CuBr and BCA Absorbance was read at 562 nm. Sample taken from column runs. Total copper was measured by atomic adsorption at 324.8 nm (0.7 mM slit).  Hydrogen Peroxide Assay Blank was phosphate buffer Standard curve using known amounts of hydrogen peroxide in chromatography buffer A. Samples from the column fractions were injected into the instrument and a reading recorded. Amount was determined from standard curve.

Results Protein damage in solution, incubation over 90 minutes Protein damage on Cu2+-IDA column, samples taken after chromatography runs

Results Effect of ascorbate on LDH inactivation in the presence of peroxide Effect of peroxide on LDH inactivation in the presence of ascorbate

Results Hydroxyl radical formation on a Cu2+-IDA column in the presence of ascorbate and or peroxide Role of oxygen and Cu+ in ascorbate mediated LDH inactivation during Cu2+-IDA column chromatography

Results

Discussion Protein Damage in Solution LDH inactivation occurs in solutions containing chelates of Cu2+ and IDA and normal levels of dissolved oxygen. Addition of ascorbate (electron donor) and peroxide enhanced the inactivation of LDH Inactivation of LDH in a solution of chelated Cu2+-IDA is due to the occurrence of a MCO reaction. Protein damage on Cu2+-IDA column In the absence of agents that promote MCO, LDH activity decreases slightly during Cu2+-IDA chromatography. This was a 5-column volume run so there is the possibility of more inactivation with longer contact time runs (i.e. at a larger scale). The requirements for metal, and the enhancement of activity loss by the presence of the reducing agent (ascorbate) and peroxide indicated the possibility that MCO reactions are occurring in the IMAC column.

Discussion Effect of ascorbate and hydrogen peroxide on Cu2+-IDA columns Low concentrations of ascorbate 1-10 mM in the presence of 2 mM peroxide cause protein loss.   High levels of peroxide are known to reduce MCO damage. This doesn’t happen in the range tested here, 1-10 mM MCO reactions during IMAC: Presence of hydroxyl radicals An MCO reaction which includes the Fenton Reaction gives rise to oxidizing species such as OH radical in the reaction between the metal and the peroxide. The presence of OH radicals indicates that a Fenton reaction (or some other radical generating reaction) occurs on the Cu2+-IDA column during chromatography in the presence of ascorbate. The OH radicals are generated not only by the conversion of the added peroxide but also by an endogenous oxygen source which is most likely the oxygen dissolved in the buffers.

Discussion Effect of other reducing agents on protein activity MCO reactions during IMAC: Partial anaerobic condition Bubbling nitrogen into the buffers produced a partial anaerobic condition in the buffers. Depleting the oxygen levels improved the recovery of activity in LDH. It’s known that anaerobic conditions prevent oxidative damage in MCO solution reactions (Shiner E et al. 1983. J. Biol. Chem. 258, 14778-14783.; Alonso-Llamazares, A M et al. Biochem. Int. 1992. 27, 879-889.). BCA prevents MCO damage to protein by complexing with Cu+ and preventing it from participating in the Fenton reaction. This indicates that Cu+ is needed for LDH inactivation on the column. Effect of other reducing agents on protein activity Protein damage is not limited to ascorbate as the electron donor in the MCO reaction; other reducing agents can cause protein damage as well.

Conclusion The requirement of oxygen, and Cu+ for LDH inactivation and the generation of hydroxyl radicals indicate that a metal catalyzed reaction of the Fenton type occurred on the Cu2+-IDA column. The hypothesis is supported. Implications: Protein damage can occur due to MCO in the earliest stages of a process but may not be evident right away Crude cell culture fluids or cell lysates contain all reagents necessary for MCO reactions on IMAC columns Characterization of other proteins on IMAC columns is needed