Download Protein damage on Cu2+

Presentation of: Conditions Promoting Metal
Catalyzed Oxidations during Immobilized CuIminodiacetic 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
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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
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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.
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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
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Stadman, E. R. Free Rad. Biol. Med. 1990, 9, 315-325
Literature Review
• Porath says: can’t compare IMAC and solutions of chelator-metalprotein and MCO reaction don’t occur on columns
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(Porath, J. Protein Expression Purif. 1992. 3, 263-281).
• These authors: MCO reactions should occur on IMAC columns
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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
– 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
• 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
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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
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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
• 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