Biotechnol. Prog. 1995, 11, 643-650
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Conditions Promoting Metal-Catalyzed Oxidations during Immobilized Cu-Iminodiacetic Acid Metal Affinity Chromatography Rajesh Krishnamurthy, Rapti D. Madurawe, Kristen D. Bush, and Janice A Lumpkin" Department of Chemical and Biochemical Engineering, ECS Building, University of Maryland Baltimore County, Baltimore, Maryland 21228
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 t o 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 t o 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 a s 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. A n understanding of the conditions under which MCO reactions occur during IMAC would aid the design of better downstream processing operations utilizing metal affinity methods.
Introduction The affinities of amino acids for metal ions were first used as a basis for the selective adsorption and elution of proteins by Porath et al. (1975). Since its first introduction, metal-affinity separations such as immobilized metal affinity chromatography (IMAC) have increasingly become accepted as a standard procedure in protein separations. IMAC has been used to purify a variety of proteins (Beitle and Attai, 1992) and has also been used in large-scale purifications (Zawitowska et al., 1992; Hochuli, 1992). In many of the examples compiled by Beitle and Attai, IMAC has been used as an initial step in fractionation and has in several instances resulted in a one-step purification. Some of the advantages attributed to IMAC are low cost, mild operating conditions, stability of the metal chelate, and high loading capacity (Arnold, 1991). General principles and mechanisms of metal-affinity separations have been well documented (see Arnold (1991) and Porath (1992) for review articles). The metal ligands most often used in IMAC are transition metals such as Cu2+,Zn2+,Fe3+,and '+
Author to whom correspondence should be addressed. 8756-7938/95/3011-0643$09.00/0
Ni2+. The chelate most widely used to immobilize metal is iminodiacetic acid (IDA), although other chelates such as nitrilotriacetic acid (NTA) and tris(carboxymethy1)ethylenediamine (TED) are also used. The amino acids that are pivotal for the coordination of transition metals have been found to be histidine and cysteine (Yip et al., 1989; Belew and Porath, 1990). It has been well established that oxygen free radicals damage biological molecules such as proteins, lipids, and nucleic acids (Swallow, 1960). An important pathway for the generation of oxygen radicals by nonradiation means is enzymatic and non-enzymatic metal-catalyzed oxidation (MCO). MCO reactions are believed to play an important role in physiological processes such as protein turnover and aging (Levine et al., 1981; Stadtman, 1992). These reactions occur in the presence of oxygen, transition metals (iron and copper, in particular), and an appropriate electron donor and convert oxygen to hydrogen peroxide (HzOz) and transition metal (M) to its reduced form (Miller et al., 1990; Halliwell and Gutteridge, 1984; Stadtman, 1990). The resultant peroxide reacts with the reduced metal to form a highly reactive oxidative species. This reaction is commonly referred to
0 1995 American Chemical Society and American Institute of Chemical Engineers
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as the Fenton reaction. The hydroxyl radical has often been shown (and is generally accepted) to be the oxidative product formed by the Fenton reaction (Gunther et al., 1995; Buettner, 1987):
+
-
Mn+ H202 *OH+ OH-
+ M(n+l)+
(1)
There is also evidence that the oxidative product formed by the Fenton reaction is not the hydroxyl radical, but a high-valence metal form (Rush and Koppenol, 1986; Rahhal and Richter, 1988). Although this ambiguity has not yet been resolved, Yamazaki and Piette (1990, 1991) have shown that factors such as the presence and nature of chelators and the concentrations of metal and peroxide play a role in determining the identity of the oxidative species formed in the Fenton reaction. It is believed that, in the case of proteins, MCO reactions occur at metal binding sites on the protein and the oxidative product (whatever its identity) attacks the side chains of amino acids a t that site (Fucci et al., 1983; Shinar et al., 1983; Levine et al., 1981). Amino acids that are most susceptible to MCO reactions are histidine, lysine, cysteine, proline, arginine, and methionine (Amici et al., 1989; Stadtman, 1990; Schoenich et al., 1993). Tryptophan, tyrosine, histidine, and cysteine have also been found to be extremely sensitive to oxidation by hydroxyl radicals generated by 6oCoradiation (Davies et al., 1987). Protein damage due to MCO reactions is often measurable in a variety of ways such as activity losses, fragmentation, cross-linking, aggregation, or increased susceptibility to proteolysis (Stadtman, 1990). The occurrence of MCO reactions in IMAC systems has not been reported. However, MCO reactions are known to occur in some solutions containing chelated metal with subsequent protein and amino acid damage (Dean and Nicholson, 1994; Stadtman and Berlett, 1991). There are several differences between metal complexes in free solution and in immobilized systems (Porath, 19921, and direct comparisons between the two are not always possible. Given the large body of literature that documents MCO reactions and related protein damage in solutions, the occurrence of these reactions in immobilized metal systems (particularly IMAC) is worthy of more investigation. There is a further compelling reason to explore these reactions as the very same amino acids that are required for metal binding during IMAC (namely histidine and cysteine) are known to be oxidized by hydroxyl radicals and are particularly sensitive toward MCO damage. The close interaction between these residues and metal during IMAC has the potential to make them prime target sites for damage in the event of the occurrence of MCO reactions. This research begins to investigate the occurrence of MCO reactions during IMAC by documenting the activity of the enzyme lactate dehydrogenase (LDH) under several initial chromatography conditions. LDH was chosen as the model protein since it is known to lose activity in MCO systems (Alonso-Llamazares et al., 1992) and has the added advantage of being a ubiquitous metabolic enzyme with a rapid and reliable activity assay. Chromatography studies were conducted on immobilized Cu2--IDA as it is by far the most commonly used IMAC system. In addition, the participation of copper in MCO reactions is well documented. The loss of activity of LDH and the formation of hydroxyl radicals on the column were studied under a variety of initial loading conditions containing dissolved oxygen or hydrogen peroxide and reducing agents (primarily ascorbate and glutathione) as Levine (1983)has shown that a model system containing trace metal, ascorbate, and oxygen (or metal and hydro-
gen peroxide) mimics enzymatic MCO systems. The addition of these reagents can also be of relevance to bioprocesses as thiol reducing agents such as dithiothreitol, glutathione, and P-mercaptoethanol in the millimolar range are often used for refolding and solubilization of inclusion bodies (Sarmientos et al., 19891, while oxygen is ubiquitously present in most processes. Crudes from cell culture may also contain high levels of dissolved oxygen and other cellular reducing agents. Since the rugged nature of IMAC makes it highly desirable for use during the initial stages of a purification protocol, the likelihood of column contact with crudes containing such reagents may be significant. In addition to the above studies, the occurrence of the Fenton reaction in the column was investigated. Protective measures are also described.
Materials and Methods Materials. A HiTrap chelating metal affinity column with IDA as the chelating group (5 mL) was obtained from Pharmacia, Piscataway, NJ. L-Ascorbic acid sodium salt (ascorbate, AH-), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), Fast Blue BB salt (FBBB), and iron(I1) sulfate heptahydrate were obtained from Aldrich, Milwaukee, WI. Chelex-100 was obtained from Bio-Rad Laboratories, Richmond, CA. Methanesulfinic acid sodium salt (MSA) was obtained from Fairfield Chemical Co., Blythewood, SC. YSI 2700 blank membranes, butanol, hydrochloric acid (concentrated), 30% hydrogen peroxide, potassium phosphate (dibasic and monobasic), copper(I1) sulfate pentahydrate, sulfuric acid (concentrated), and toluene were obtained from Fisher Scientific (Pittsburgh, PA). Dimethyl sulfoxide (DMSO, Hybri-Max), bicinchoninic acid (BCA), iminodiacetic acid, and L-lactic dehydrogenase Type XI from rabbit muscle were obtained from Sigma, St. Louis, MO. All reagents were A.C.S. grade unless otherwise specified. Equipment. An FPLC unit with a LCC-501 Plus controller from Pharmacia, Piscataway, NJ, was used for the chromatography runs. LDH activity assays and protein determinations were done on an EL 340 biokinetics reader from BIO-TEK Instruments, Winooski, VT. A UV 2101 PC spectrophotometer from Shimadzu Scientific Instruments Inc., Columbia, MD, was used for the Cu+ measurements and GSH assays. Other spectrophotometric measurements were done on an HP 8452A diode array spectrophotometer from Hewlett Packard, Palo Alto, CA. Hydrogen peroxide concentrations were determined with a YSI model 2700 glucose analyzer, Yellow Springs Instruments, Yellow Springs, OH. Total copper analyses were done on a Perkin-Elmer 4200ZL atomic absorption spectrometer. Chromatography Procedure. All buffers used for chromatography procedures and sample preparations were treated with Chelex-100 to remove trace amounts of metal contaminants. The column was loaded with 1 mL of 100 mM CuSO4 and washed with 5 column volumes of 12 mM sodium acetate, pH 4.0, to remove loosely bound copper. The actual copper capacity of the column used is 23 pmollmL of gel, and the column was only 80% saturated with copper in order to leave a zone of unchelated IDA a t the end to trap copper ions that may be leached during chromatography. The column was equilibrated in 0.5 M NaCV20 mM sodium phosphate, pH 7 (buffer A). The elution buffer was 50 mM imidazole/ 0.5 M NaCU20 mM sodium phosphate, pH 7 (buffer B). The column was stripped with 68 mM EDTA after five chromatographic runs and reloaded with fresh metal as described above. A blank run was carried out after each new metal loading. The chromatographic runs were conducted in duplicate a t room temperature. The flow
Biotechnol. frog., 1995,Vol. 11, No. 6
rate was 1mumin. The sample loop, needle and syringe were thoroughly rinsed with deionized water to prevent metal contamination prior to contact with the Cu2+-IDA column. All stock solutions were made in buffer A. For each run, LDH, buffer A, and either hydrogen peroxide, reducing agent, or both were mixed in a total volume of 1.0 mL to get the desired final concentrations. Unless otherwise stated, the final concentration of LDH was 2.0 mg/mL. A 0.2 mL aliquot of this was injected onto the Cu2+-IDA column. The remainder was stored in an aluminum foil wrapped tube for activity and protein determinations (the control sample). The protein was eluted with buffer B using a step gradient. A 0.5 mL amount of 68 mM EDTA in buffer A was added to all fraction collection tubes prior to sample collection in order to stop MCO reactions from occurring after the chromatography step. Elution fractions of 3 mL were collected in these tubes, vortexed, and stored on ice. These fractions and the control were assayed for activity at the end of the run. Solution Studies. The final concentrations of the incubates were 10 nM LDH, 0.04 mM CuS01, 0.4 mM IDA, 0.4 mM BCA, 2 mM ascorbate, and/or 2 mM peroxide in 80 mM sodium phosphate buffer, pH 7.0. For the activity assays, LDH was incubated at room temperature in solutions containing one or more of the other reagents or in phosphate buffer without any additions (control). At a given sampling time, a 0.1 mL aliquot was assayed for activity as given below. The activities are given as a percentage of the activity of the control LDH sample in phosphate buffer. For the hydroxyl radical measurements, solutions containing one or more of the above reagents were incubated with 0.02 mL of pure DMSO in a final volume of 1.0 mL. At a given sampling time, a 0.2 mL aliquot was quenched with 1.8 mL of 10 mM EDTA and assayed for hydroxyl radicals as given below. LDH Activity Assay. The substrate for the LDH activity assay contained 1mM sodium pyruvate and 0.22 mM NADH in 0.2 M tris-HC1, pH 7 (Worthington, 1988). For solution studies, 0.1 mL of enzyme solution was added to a quartz cuvet containing 3 mL of substrate and the initial decrease in absorbance at 340 nM wavelength over time was measured on a HP spectrophotometer. For the column studies, 0.18 mL of the substrate was added to each well of a microtiter plate. Aliquots of the initial sample (0.05 mL) and the column elution fractions (appropriately diluted in buffer A to be within the range . of the assay) were added to the microtiter plate wells to get a total reaction volume of 0.23 mL. The decrease in absorbance at 340 nm wavelength was monitored using the biokinetics reader. All assays were carried out in duplicate. The activity of LDH was defined as units/mL = (-AA,,Jmin) x (sample volume/reaction volume)(sample dilution)
Protein Determination. Protein determinations of the initial sample and the elution fractions were done in duplicate according to the Peterson's modification of the micro-Lowry method as given by procedure number 5656 of the protein assay kit from Sigma Diagnostics. The protein concentrations of the samples were calculated in milligrandmilliliter units using a standard curve constructed with k n o w n amounts of bovine serum albumin. Typically, only two of the fraction tubes were found to contain protein. The total protein recovered after the chromatography step was taken as the sum of these values. The percentage of protein recovered was calculated on the basis of the amount of protein injected onto the column. These recoveries were typically 85-95%.
645
Specific Activity. The specific activity (unitdmg of protein) was calculated as the ratio of activity (unitdmL) to protein concentration (mg/mL). The specific activities of the two elution fractions containing protein were first calculated for each fraction and were found to be very similar. These values were averaged to obtain the specific activity of LDH recovered after chromatography. For easy comparison of the extent of LDH damage, this value was further normalized to the specific activity of the initial LDH sample (control) after an incubation period corresponding to the chromatography step. Unless otherwise specified, all specific activity data given are normalized as described above. Hydroxyl Radical Assay. For column studies, 0.2 mL of pure DMSO, 0.08 mL of hydrogen peroxide or reducing agent, and 0.72 mL of buffer A were mixed and 0.5 mL was immediately injected onto the Cu2+-IDA column. Fractions were collected in EDTA filled tubes as described above. Cumulative *OH concentrations were determined by trapping 'OH with dimethyl sulfoxide (DMSO) and measuring the primary product, methanesulfinic acid (MSA), by the colorimetric assay described by Babbs and Griffin (1989). Two minor modifications to the original protocol were made. First, in order to eliminate interference from unreacted ascorbate, the pH of a 0.2 mL aliquot of the elution fraction was lowered to 1.5 by adding HzS04 prior to assaying for MSA. Second, the pyridine addition step used in the original protocol to stabilize the color compound was omitted as the final color compound was reasonably stable over a period of 15 min under our conditions. The absorbance of the final extract containing MSA was measured at 424 nm against a blank, which was prepared with deionized water and carried through the same colorimetric procedure. The concentration was determined by relating the absorbance values to a standard curve, which was constructed with k n o w n amounts of pure MSA in phosphate buffer. Copper Determinations. Cu+ Content. A 0.5 mL aliquot of a sample mixture containing 0.6 mM bicinchoninic acid (BCA) and either ascorbate, peroxide, or both was injected onto the column. Chromatography and sample collection were as previously described, except that no EDTA was added to the fraction tubes. A bright purple color was indicative of the formation of a BCACu+ complex. This complex was quantified by measuring the absorbance of the elution fractions at 562 nm wavelength on a SHIMADZU UV-vis spectrophotometer. The concentration of Cu+ was determined by relating the absorbance values to a standard curve, which was constructed with known amounts of CuBr and BCA. Total Copper. Total copper contents of the elution fractions were determined at 324.8 nm wavelength and 0.7 mM slit width using a Perkin-Elmer 4200ZL atomic absorption spectrometer. Hydrogen Peroxide Assay. Hydrogen peroxide levels were determined with a YSI 2700 glucose analyzer using a blank membrane. The YSI analyzer was calibrated to read 0 for phosphate buffer (no peroxide), and a standard curve was constructed using k n o w n amounts of hydrogen peroxide in buffer A. At a sampling time, an aliquot of the column elution fractions was injected into the YSI 2700 analyzer, and the reading was recorded. Results and Discussion Protein Damage in Solution. Experiments were first conducted in solutions containing Cu2+-IDA chelates to establish whether metal catalyzed oxidation reactions can occur with this metal-chelator pair. Figure 1 shows the effect on LDH activity when 2 mM ascorbate, 2 mM hydrogen peroxide or a mixture of both
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loo
0
1
IDAonly IDA.Cu" IDA, cu2'. peroxide
IDA. Cu". ascorhatr
Y
60
0 IDA. Cu". peroxide, ascorbate
I 2
€3
0
0 0
20
40
60
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1
No Addrtion
Hi02
Ascothale
Ascorbate "202
Time (min) Figure 1. Loss of LDH activity in solutions containing Cu2+, IDA, ascorbate, and hydrogen peroxide. Activity is given as a percentage of the LDH activity in buffer alone (control).The mM LDH, 0.04 mM Cu2+,0.4 mM final concentrations are IDA, 2 mM ascorbate, and 2 mM peroxide in 80 mM sodium phosphate buffer, pH 7.0. The incubations were at room tem-
perature.
was added to a solution containing 0.04 mM Cup+,0.4 mM IDA, and 10 nM LDH. A 10-fold excess of IDA was added to ensure that all metal in the system was chelated. The activity of LDH is represented as a percentage of the activity of the control sample of LDH in buffer. In the absence of either added ascorbate or added peroxide, activity of LDH in a Cu2'-IDA solution decreased to 30% compared to that of the control over a time period of 90 min (filled circles). With the addition of ascorbate and peroxide, the loss of LDH activity was almost instantaneous and complete (open squares). Complete inactivation was also observed with the addition of ascorbate alone to the Cu2+-IDA solution (filled diamonds) while the addition of peroxide alone resulted in a slightly slower rate of inactivation (filled triangles j. The presence of copper was essential for LDH inactivation as IDA alone did not affect activity (open circles). Similarly, in the absence of copper, ascorbate alone and peroxide alone did not affect LDH activity over the time period tested (data not shown). The data represented here show that protein inactivation occurred in solutions containing chelates of Cu2+IDA and normal levels of dissolved oxygen and that addition of ascorbate and peroxide enhanced the inactivation. Stadtman (1990) documents several types of nonenzymatic MCO systems containing a transition metal, reducing agent, oxygen, or hydrogen peroxide that have been used to study protein modifications. Loss of enzymatic activity is a common characteristic of protein damage in these systems (Levine, 1983; Shinar et al., 1983) where ascorbate promotes MCO reactions by reducing metal while the addition of peroxide enhances radical generation. Similarly, it has been shown that other solutions containing chelates of copper such as Cu2--phenanthroline complexes undergo MCO reactions resulting in hydroxyl radical generation (Florence, 1984) and DNA degradation (Gutteridge and Halliwell, 1982). Therefore, it can be concluded that the inactivation of LDH was due to the occurrence of MCO reactions in solutions of chelated Cu2--IDA. As the copper in an IMAC system is not attached through covalent links, but through coordination bonds to the immobilized IDA, the occurrence of MCO reactions on Cu2+-IDA columns (similar to that observed for Cu2--IDA solutions) appears to be highly probable, even though there are several differences (such as steric considerations j between the two systems.
Figure 2. Loss of LDH activity after chromatography on a Cu2+-IDAcolumn. A 0.2 mL sample of 2 mgimL LDH containing either 2 mM hydrogen peroxide, 2 mM ascorbate, or 2 mM each of ascorbate and peroxide in buffer A was loaded onto the column and eluted with a step gradient of buffer B. EDTA was added to the elution fractions to terminate any postcolumn MCO reactions. The fractions were assayed for LDH activity and protein content, and the specific activity was calculated as given in Materials and Methods. The data are represented as a percentage of the specific activity of the corresponding control containing the same added reagents but had not undergone
Cu2+-IDA chromatography.
Protein Damage on Cu2'-IDA Columns. To determine whether a loss of activity occurs in immobilized Cup+-IDA columns, 2 mg/mL of LDH was injected onto a Cup+-IDA column in the presence or absence of added hydrogen peroxide (2 mM) and ascorbate (2 mM). The retention time of LDH on the column was approximately 24 min for each of the chromatography runs and did not vary under the different load conditions tested. The protein eluted in a single peak for all load conditions and was recovered in two elution fractions. Both fractions had similar specific activities indicating that the protein eluted as a single pool. Figure 2 lists the specific activity of the protein recovered after chromatography on the Cu2'-IDA column. For convenient comparison, all specific activities recovered after chromatography were normalized to those of the initial LDH samples that were injected onto the column (controls). Each control therefore contained the same added reagent as that of the corresponding chromatographic run. The activities of the controls were taken after a time duration corresponding to that of the chromatography run. Therefore, only the activity changes due to passage through the Cup'-IDA column are reflected by the data. Comparison of the specific activity also minimized differences caused by slight variations in protein recoveries. Although a small loss of specific activity was observed when only LDH was injected onto the Cu2--IDA column, it was concluded that little or no enzyme inactivation occurred as these losses were within the margin of recovery errors. However, the specific activity decreased t o 75% when the chromatography was done in the presence of 2 mM hydrogen peroxide alone and to 73% in the presence of 2 mM ascorbate alone. Specific activity loss was highest when both ascorbate and peroxide (2 mM each) were present with only 38% being recovered. Contact with immobilized Cu2+-IDA was essential for the observed loss of LDH activity in the presence of ascorbate and/or peroxide. The requirement for metal and the enhancement of protein damage by the reducing agent and peroxide indicated the possibility that MCO reactions occurred in this immobilized system. The activity losses observed on the column were not as dramatic as that observed in solution. Lower levels of protein damage observed for the immobilized system may be due to a
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Figure 3. Effect of ascorbate on LDH inactivation during Cu2+-IDAcolumn chromatography in the presence of hydrogen peroxide. A 0.2 mL sample containing LDH (2 mg/mL),hydrogen peroxide (2 mM), and varying concentrations of ascorbate was loaded onto the column. The experimental details and specific activity calculations are as given in Figure 2. The data are represented as a percentage of the corresponding control as described previously.
variety of factors such as differences between metal complexes in free solution and in immobilized systems, experimental variations such as the higher LDH concentration, and shorter contact times due to different rates of mobility (and therefore separation) among LDH, ascorbate, peroxide, etc. Although in the absence of added agents no loss of activity was detected after chromatography on Cu2+-IDA, data obtained for Cu2+IDA solutions may indicate the possibility of more significant levels of damage with longer chromatography (contact) times. Effect of Ascorbate and Hydrogen Peroxide on Cu2+-IDA Columns. In order to assess the effect of ascorbate on the column, varying amounts of ascorbate in the presence of 2 mM hydrogen peroxide were injected with LDH (2 mg/mL) onto the Cu2+-IDA column. As indicated in Figure 3, low concentrations of ascorbate (1 mM) are capable of causing protein damage. Nearly 80% of the protein specific activity was lost above ascorbate concentrations of 10 mM. High levels of ascorbate (200 mM) were not able to reverse protein damage. Protection at high ascorbate was expected as, in addition to its prooxidant role, ascorbate can also act as an antioxidant by scavenging radicals (Yamamoto et al., 1987). The relative importance of this dual function is believed to be dependent not only on the concentration of ascorbate but also on the concentration, form (reduced or oxidized),and structure of the metal ions. The amount of copper in the column (1 mL x 100 mM = 0.100 mmol) is far greater than the highest amount of ascorbate present (200 mM x 0.2mL = 0.040 mmol). Therefore, even at the highest concentration used, the amount of ascorbate present was not sufficiently in excess to offer protection from inactivation. The effect of varying amounts of peroxide on LDH activity during chromatography on Cu2+-IDA in the presence of 1 mM ascorbate is given in Figure 4. With increasing concentrations of peroxide, the specific activity decreased to 40% of the control value over the concentration tested. Several studies in MCO solutions have shown that high levels of peroxide are able to reverse MCO-related damage and lower hydroxyl radical levels (Levine, 1983; Florence, 1984). Although the mechanism of this protection is not fully understood, it is believed that at high concentrations peroxide acts as a radical scavenger and/or helps maintain metal in the higher valence form, thereby reducing MCO-related damage. The levels of peroxide present here (1-10 mM) were most likely not high enough to offer protection from protein inactivation. MCO Reactions during IMAC. Presence of Hydroxyl Radicals. The reaction between peroxide and
I
2
4
6
6
1
Peroxide Concentration (mM)
0
Figure 4. Effect of hydrogen peroxide on LDH inactivation during Cu2+-IDA column chromatography in the presence of ascorbate. A 0.2 mL sample containing 2 mg/mL of LDH, 1mM ascorbate, and varying concentrations of hydrogen peroxide was
loaded onto the column. The experimental details and specific activity calculations are as given in Figure 2. 1507
"
I
no addition
H202
Ascorbate
Ascorbate
H202
Figure 6. Hydroxyl radical formation on a Cu2+-IDAcolumn in the presence of ascorbate and/or peroxide. A 0.5 mL sample containing an excess of DMSO and either ascorbate (1 mM), hydrogen peroxide (0.2 mM), or both ascorbate and peroxide was
injected onto the column and eluted as described previously. The elution fractions were assayed for MSA as given in Materials and Methods. The amount of hydroxyl radicals generated is measured as the amount of MSA present in the elution fractions. metal (Fenton reaction) gives rise to oxidizing species such as the hydroxyl radical or a high-valence metal form. In order to investigate the possibility of the Fenton reaction occurring in immobilized systems, experiments were conducted to determine whether hydroxyl radicals were formed during chromatography with Cu2+-IDA. Hydroxyl radicals can be conveniently determined by the spectrophotometric assay where the hydroxyl radical scavenger, dimethyl sulfoxide (DMSO), is used to react with the radicals, and the resulting non-radical product, methanesulfinic acid (MSA), is colorimetrically determined after derivatization with a diazonium salt (Babbs and Griffin, 1989). As stated previously, there is some ambiguity regarding the identity of the oxidative species generated by the Fenton reaction. The conversion of DMSO to MSA indicates the presence of an oxidative species. Given that literature details only the scavenging of hydroxyl radicals by DMSO and not the scavenging of high-valence copper forms, this oxidative product is assumed to be the hydroxyl radical. Figure 5 shows the amount of hydroxyl radicals detected when 0.5 mL of an aliquot containing excess DMSO and either 0.2 mM hydrogen peroxide, 1mM ascorbate, or a mixture of both was injected onto the Cu2+-IDA column. In the presence of both peroxide and ascorbate, 143 nmol of hydroxyl radicals was trapped by DMSO. In the presence of ascorbate alone, 109 nmol of hydroxyl radicals was detected while only 7 nmol was detected in the presence
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of peroxide alone. In the absence of any additives, no radicals were detected. Due to the mobility of DMSO through the column, the amount of radicals measured here most likely reflects only a portion of the total radicals generated in the column. The results shown here are similar to a number of studies where solutions of copper and complexes of copper (notably phenanthroline complexes) have been shown to promote the formation of hydroxyl radicals via MCO reactions (Florence, 1984; Rowley and Halliwell, 1983; Ueda et al., 1994). The presence of hydroxyl radicals indicates that the Fenton reaction, or a similar radical-generating MCO reaction, occurred on the Cu2+-IDA column during chromatography in the presence of ascorbate. The data also indicate that hydroxyl radicals were generated not only by the conversion of added hydrogen peroxide but also by an endogenous oxygen source which is most likely to be the dissolved oxygen present in the buffers. The different levels of hydroxyl radicals generated during chromatography in the presence of ascorbate, peroxide, or both suggest that the mechanisms of protein inactivation are different for these three cases. Florence (1984) has outlined three different mechanisms (all of which pertain to MCO reactions) for solutions of Cu2+-phenanthroline in the presence of glutathione (GSH) and hydrogen peroxide, GSH and oxygen, and peroxide alone. In the first two cases, hydroxyl radicals were postulated to be generated either directly by the Fenton reaction or indirectly through an oxygen-derived superoxide intermediate, upon reduction of Cu2+by GSH. In the third case, the reaction of hydrogen peroxide and Cu2+ was postulated to yield superoxide and Cu+. Although this furthers our understanding of MCO reactions, any mechanistic parallels between the free solution and immobilized systems remain highly speculative at this stage. The results of Ueda et al. (1994) are also of interest as they demonstrate through electron spin resonance (ESR)-spin trapping techniques that copper(11) complexes with oligopeptides containing histidines converted hydrogen peroxide to hydroxyl radicals. Dissolved Oxygen. The role of oxygen in the ascorbate-mediated inactivation (in the absence of peroxide) was further investigated by bubbling nitrogen through the buffers during chromatography. Although this method most likely did not result in complete anaerobic conditions, at least partial anaerobic conditions were achieved. As indicated by Figure 6, in the presence of nitrogen, the amount of protein damage decreased for all three ascorbate concentrations tested. Full protection was not observed most likely due to incomplete anoxia. Similar to these findings on Cu2+-IDA columns, it has been shown that anaerobic conditions prevent oxidative damage in MCO solutions (Shinar et al., 1983; AlonsoLlamazares et al., 1992). Reaction with Cu+. The occurrence of the Fenton reaction is one of the key radical generating-reactions in MCO systems, and oxidations caused by radicals are responsible for MCO damage of proteins (Stadtman, 1990). When LDH was incubated in a solution of copper, ascorbate, and peroxide for 30 min, nearly all of its activity was lost while complete recovery was obtained in the presence of the Cu+ specific chelator bicinchoninic acid (BCA) (data not shown). BCA prevents MCO damage of proteins by complexing Cu+ and preventing its participation in the Fenton reaction. To further investigate whether the occurrence of the Fenton reaction during chromatography on the Cu2+-IDA column is responsible for the observed loss of protein activity, 2 mgl mL of LDH, 10 mM ascorbate, and 2 mM peroxide were injected onto the column in the presence or absence of 0.6 mM BCA. In the absence of BCA, the specific activity
100-
80-
.-x Y
. YI
2
With 2 m M H202 Ijo-
Without YO, Without H202. with N, With 2 m M H202, with BCA
1
10
100
Ascorbate Concentration (mM) Figure 6. Role of oxygen and Cu+ in ascorbate-mediated LDH inactivation during Cu2+-IDA column chromatography. All chromatography runs contained 2 mg/mL of LDH and ascorbate (1,10, or 100 mM). The runs with 2 mM hydrogen peroxide in the loading sample were carried out either in the presence or absence of 0.6 mM BCA. The peroxide-containing runs were performed with buffers containing normal amounts of dissolved oxygen (i.e., no added oxygen). The runs without peroxide were performed either with buffers containing normal amounts of dissolved oxygen or with buffers where nitrogen was bubbled through to obtain partial anoxia. Table 1. Effect of Thiol Reducing Agents on LDH Activity during Chromatographv on Cu2+-IDAColumns % LDH activity
0.1 mM GSH 1mM GSH 10 mM GSH
5 mM cysteine 5 mM P-mercaptoethanol
45 26 8 32 35
after chromatography decreased to 36% (Figure 6). In contrast, the presence of BCA reduced LDH inactivation, indicating that the participation of Cu+ was necessary for protein damage. The requirement of oxygen and Cu+ for LDH inactivation and the generation of hydroxyl radicals by LDH inactivation indicated that the participation of Cu+ was necessary for protein damage. The requirement of oxygen and Cu+ for LDH inactivation and the generation of hydroxyl radicals indicate that a metalcatalyzed reaction of the Fenton type occurred on Cu2+IDA columns. Thiol Reducing Agents. To determine whether or not metal-catalyzed damage is specific to the ascorbate/ peroxide system, varying amounts of GSH were injected to the column. The results given in Table 1indicate that the observed protein damage was not limited to ascorbate only but that GSH was also capable of inactivating protein in an IMAC column, with increasing concentrations giving rise to increased protein damage. Similarly, other thiol agents like cysteine and P-mercaptoethanol also lowered the activity of LDH during Cu2+-IDA chromatography. Loss of Copper from the IMAC Column. To evaluate loss of copper from the column, chromatography runs were conducted in the presence of 0.6 mM BCA with added reducing agent and/or hydrogen peroxide. BCA was included in these runs because the formation of a deep purple color upon complexation with BCA indicates the presence of Cu+. The Cu+ content of the elution fractions can therefore be quantitatively determined by measuring the absorbance of this complex at 562 nm wavelength. Elution fractions were also analyzed for total copper content (Cu2+and Cu+)by atomic absorption spectroscopy. The results are shown in Table 2. Experiments were first conducted to verify that the presence of BCA did not affect the outcome of the copper removal
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Table 2. Removal of Copper during IMAC on Cu2+-IDA Columns total copper &mol) BCA only LDH only BCA + 2 mM ascorbate BCA + 2 mM H202 BCA + 2 mM ascorbate, 2 mM H202 BCA 5 mM cysteine BCA 2 mM GSHa BCA 1mg/mL of Gly-His-Lys
+ + +
a
Cu+ &mol)
0 0 0.490
0 0
0.171
0.1 0.4 0.6
0.406
0.732 0.178 0.258
0.5
0.1 0
0.2 mL loaded onto column.
from the column, and it was indeed found not to remove copper from the column. The addition of a reducing agent or hydrogen peroxide resulted in the removal of copper from the column. This loss of copper occurred in spite of providing a zone of unchelated IDA on the column for the capture of any leached metal (Beitle and Attai, 1992). In the presence of ascorbate, nearly all of the copper was removed from the column in the form of Cu+. The thiol reducing agents cysteine and GSH also removed copper in the reduced Cu+form. The amount of copper removed was dependent not only on the type of reducing agent but also on the concentration, with higher concentrations resulting in higher copper loss (data not shown). These results indicate that chromatography in the presence of reducing agents resulted in the removal of copper mainly due to the reduction of immobilized Cu2+by the reducing agents. The copper removed in the presence of peroxide was present as both Cu2+and Cu+. This may well be due to the reduction of Cu2+by hydrogen peroxide as proposed by Florence (1984). In contrast to the above data, the copper removed by the tripeptide glycine-histidine-lysine (GHK) was present only in the Cu2+form. Belew and Porath (1990) have observed the lack of retention of GHK on a Cu2+-IDA column and have proposed that this is due to metal ion transfer. The removal of copper as Cu2+ supports this theory. The ratio of GHK to Cu2+appears to be higher (approximately 6:l) than expected for a GHK-Cu2+ complex presumably due to capture of some of the leached Cu2+by the free IDA zone of the column. In the presence of reducing agents or peroxide, loss of copper occurred after 5 min of the chromatography procedure whereas LDH is eluted after 24 min. Therefore, in the presence of these agents, LDH fractions are not likely to be significantly contaminated with Cu2+due to the difference in mobility. In addition, the loss of metal is very low compared to the copper capacity of the column. Similarly, Hochuli (1988) has reported only minimal levels of copper contamination of protein (30-50 ng/mg of protein) during the purification of interferon a-2a on Cu2+-IDA columns. The impact of metal loss on IMAC procedures that do not contain reductants or peroxide also appears to be minimal because chromatography of LDH alone did not result in the loss of copper.
Conclusions Little is known about the occurrence of MCO reactions on IMAC systems. Knowledge of the conditions under which these reactions occur and the forms of damage that can result are important for the design of successful purification processes that utilize IMAC. We have shown here that LDH undergoes a significant loss of activity when chromatographed on a Cu2+-IDA IMAC column in the presence of reducing agents such as ascorbate and thiol agents (GSH, cysteine, etc.). Decrease in activity losses under partial anaerobic conditions indicated that oxygen was required for enzyme inactivation. Chromatography in the presence of the oxidant hydrogen perox-
ide also resulted in activity losses. The most damage was observed in the presence of both ascorbate and peroxide, with increasing concentrations of reducing agent or peroxide leading to increased levels of damage. During chromatography in the presence of reducing agents, the immobilized Cu2+was converted to Cu+ and metal ions were lost from the column. The chelation of Cu+by BCA prevented the loss of LDH activity. The formation of hydroxyl radicals, the requirement for Cu+ and oxygen, and the stimulation of LDH inactivation by hydrogen peroxide indicated that metal-catalyzed oxidations occurred on the immobilized Cu2+-IDA column in the presence of reducing agents and/or peroxide. These results imply that Cu2+-IDA chromatography may result in a damaged protein product when using crudes containing reductants and/or oxidants. Such crudes could easily result by the addition of reducing agents for the solubilization of inclusion bodies (Sarmientos et al., 19891, through exposure to trace amounts of sanitizing agents such as peroxide (Kirsch et al., 1993) or through the maintenance of high levels of dissolved oxygen during cell culture. Crudes resulting from cell lysates may be especially vulnerable because of the presence of endogenous reducing agents and enzymatic systems that trigger MCO reactions. The occurrence of MCO reactions under the various conditions tested here point to the need for evaluation of the precolumn operations to determine that starting materials are devoid of the reagents that would result in MCO reactions upon IMAC. Under all conditions tested, the chelation of Cut by BCA. Similarly, in the presence of ascorbate, the operation of the column under anaerobic conditions minimized protein damage. These results may well provide a basis for the design of practical preventive methods to minimize protein damage during IMAC. The replacement of high redox-active Cu with a less active metal such as Zn may also prevent or at least minimize protein damage. In the absence of added reducing agent or peroxide, MCO reactions were not detected on the column over the chromatography times of this study as all of the criteria used to determine the occurrence of MCO reactions yielded negative results. However, the occurrence of such reactions in solutions indicates the possibility that, with larger columns and longer column contact times (as would be necessary for large-scale applications), discernible protein inactivations could occur. It must also be kept in mind that enzyme inactivation is but one form of overt damage caused by MCO reactions and that a variety of relatively subtle changes that are not easily measurable can also occur due to MCO reactions. For example, weakening of the subunit structure, decreased thermal stability, and changes in isoelectric point have been observed when glutamine synthetase was subjected to MCO solutions (Rivett and Levine, 1990). Therefore the overall effect of MCO reactions on proteins may not always be immediately apparent but would nevertheless result in a damaged protein exhibiting microheterogeneity. Detailed understanding of these reactions on immobilized metal systems and the identification of the reactive sites and the types of amino acid modifications that occur would confirm the mechanisms of protein damage and aid the design of better IMAC procedures that result in highly active and intact proteins.
Acknowledgment Support from the National Science Foundation Research Initiation Award (#BCS-9309569) and the UMBC Designated Research Initiative Fund are gratefully acknowledged.
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BP950059N @Abstract published in Advance ACS Abstracts, October 1, 1995.
