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Chem. Res. Toxicol. 2000, 13, 26-30
Enzymatic Reduction of Arsenic Compounds in Mammalian Systems: Reduction of Arsenate to Arsenite by Human Liver Arsenate Reductase Timothy R. Radabaugh† and H. Vasken Aposhian*,‡ Graduate Interdisciplinary Program in Genetics and Department of Molecular and Cellular Biology, The University of Arizona, Tucson, Arizona 85721 Received June 30, 1999
An arsenate (AsV) reductase has been partially purified from human liver. Its apparent molecular mass is approximately 72 kDa. The enzyme required a thiol and a heat stable cofactor for activity. The cofactor is less than 3 kDa in size. The thiol requirement can be satisfied by dithiothreitol (DTT). However, the extent of stimulation of reductase activity by glutathione, thioredoxin, or reduced lipoic acid was negligible compared to that of DTT. The heat stable cofactor does not appear to be Cu2+, Mn2+, Zn2+, Mg2+, or Ca2+. The enzyme does not reduce monomethylarsonic acid (MMAV). The isolation and characterization of this enzyme demonstrates that in humans, the reduction of arsenate to arsenite is enzymatically catalyzed and is not solely the result of chemical reduction by glutathione as has been proposed in the past.
Introduction The putative pathway for the biotransformation of inorganic arsenic in mammals has suggested that inorganic arsenate (AsV)1 is reduced to inorganic arsenite (AsIII) in the blood (1, 2) which then in the tissues is either sequestered by arsenite binding proteins (3, 4) and/or enzymatically methylated (5-7) to form monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV). These urinary metabolites are present in the urine of some but not all mammals (8, 9). It has been proposed that methylation is a detoxification step as the methylated metabolites are supposedly less acutely toxic than either inorganic arsenate or arsenite. But arsenite is more toxic than arsenate since it has a higher affinity for sulfhydryl groups, including those of important enzymes such as pyruvate dehydrogenase (10). Since both protein binding and methylation of arsenic species require an oxidation state of +3, reduction of arsenate is a critical step for its biotransformation. Glutathione can reduce arsenate nonenzymatically in vitro (11, 12), and it is currently believed that mammals reduce arsenate in the blood (1, 2) before uptake into the tissues where methylation and protein binding can occur. In Escherichia coli and Staphylococcus aureus, plasmids containing the arsC gene encode an arsenate reductase that reduces arsenate in the cell to arsenite, which is then actively effluxed from the cell via an ATPdependent pump (13). The ATPase activity of the ArsA protein is not activated by arsenate, hence the need for * To whom correspondence should be addressed: Department of Molecular and Cellular Biology, P.O. Box 210106, The University of Arizona, Tucson, AZ 85721- 0106. Phone: (520) 621-7565. Fax: (520) 621-3709. E-mail:
[email protected]. † Graduate Interdisciplinary Program in Genetics. ‡ Department of Molecular and Cellular Biology. 1 Abbreviations: AsV, arsenate; AsIII, arsenite; MMAV, monomethylarsonic acid; DMAV, dimethylarsinic acid; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; MOPS, 3-(N-morpholino)propanesulfonic acid.
reduction (14). Clearly, in these two bacterial systems, reduction is part of a detoxification mechanism since arsenite is removed from the cell. Recently, a novel arsenate reductase, Arr, which plays an essential role in the growth of Chrysiogenes arsenatis has been purified and characterized (15). This arsenate reductase has properties different from those found in E. coli and S. aureus. In this case, arsenate is reduced by accepting an electron from acetate to complete the anaerobic respiratory chain and the reduction is catalyzed by Arr. Most work to date dealing with arsenate reductases has dealt with enzymes in prokaryotes; however, recently, ACR1, ACR2, and ACR3 genes of Saccharomyces cerevisiae have been found to confer resistance to arsenic (16) and the ACR2 gene has been shown to encode an arsenate reductase (17). Our objective was to determine if there is an arsenate reductase in human liver. The sequences of E. coli and S. aureus bacterial reductases are only 18% similar (14). This suggests a low level of sequence conservation for these enzymes. Therefore, we chose as our approach the development of an assay and purification procedure for arsenate reducing enzyme activity rather than using molecular cloning. This paper demonstrates that human liver has an arsenate reductase which reduces arsenate to arsenite. The arsenate reductase has an approximate molecular mass of 72 kDa and requires a dithiol and a heat stable cofactor for optimal activity.
Experimental Procedures Caution: Arsenic has been classified as a human carcinogen by the International Agency for Research on Cancer (18). Reagents. Sodium arsenate was ACS reagent grade and was purchased from MCB Reagents (Cincinnati, OH). Carrier free [73As]arsenate was purchased from Los Alamos National Laboratory (Los Alamos, NM). [14C]Methylarsonic acid (disodium salt) was purchased from ARC Inc. (St. Louis, MO). DEAESephacel, Sephacryl S-200 HR, and molecular mass standards were purchased from Pharmacia Biotech (Uppsala, Sweden). Glutathione, thioredoxin, lipoic acid (DL-6,8-thioctic acid, re-
10.1021/tx990115k CCC: $19.00 © 2000 American Chemical Society Published on Web 12/16/1999
Human Liver Arsenate Reductase
Chem. Res. Toxicol., Vol. 13, No. 1, 2000 27
Table 1. Partial Purification of Human Liver Arsenate Reductase fraction
step
volume (mL)
protein (mg/mL)
activity (unitsa/mL)
specific activity (units/mg)
recovery of units (%)
purification (x-fold)
I II III
cytosol DEAE-Sephacel Sephacryl S-200 HR
75.0 3.5 4.1
52.9 203.5 25.1
45.5 1419.0 545.0
0.9 7.0 21.7
100.0 145.6 65.5
7.7 24.1
a
A unit is defined as 1 nmol of arsenite formed per 30 min.
duced form), ovomucoid (containing ovoinhibitor), phenylmethylsulfonyl fluoride (PMSF), and catalase were obtained from Sigma Chemical Co. (St. Louis, MO). The protease inhibitor E-64 was obtained from Peptides International (Louisville, KY). Monoflow III scintillation fluid was obtained from National Diagnostics (Atlanta, GA). All other reagents were of the highest quality that could be obtained. Tissue. Human liver from a male was obtained from the Association of Human Tissue Users (Tucson, AZ). The donor did not die of a liver-related illness. Arsenate Reductase Assay. The assay reaction mixture contained 0.10 M MOPS buffer (pH 7.0), 1.0 mM EDTA, 500 µM DTT, 10 µL of 2-fold diluted cofactor preparation (not necessary for assaying fractions I and II, Table 1), 190 µM sodium [73As]arsenate (0.5 µCi, 100-150 cpm/pmol of arsenic), and the enzyme preparation. The final reaction volume was 100 µL. Reaction mixtures were incubated for 30 min at 37 °C; then 100 µL of 0.10 M sodium arsenite was rapidly added, and the reaction tubes were immersed in a boiling water bath for 3 min to end the reaction. Tubes were cooled on ice for 5 min and then centrifuged at 12000g for 12 min. A 50 µL sample of the supernatant was injected onto either a 5 mm × 250 mm PhaseSep ODS2 C18 column using 20 mM tetrabutylammonium phosphate buffer (pH 5.3) as the mobile phase with a flow rate of 1 mL min-1 or a 4.9 mm × 250 mm Hamilton PRP-X100 anion exchange column using 30 mM sodium phosphate buffer (pH 6.0) with a flow rate of 1.5 mL min-1 (19). The arsenite and arsenate were quantified by a postcolumn inline Beckman 171 radioisotope detector. Monoflow III was used as the scintillation fluid with a flow rate that was 3 times that of the mobile phase. To determine if our assay was linear with respect to protein concentration, 10, 25, 50, and 100 µg of fraction III (see below) were assayed as described above. To determine the linearity of the assay with time, 50 µg of fraction III (see below) was incubated for 10, 20, 30, 40, 50, and 60 min. At the end of each incubation, 100 µL of 0.10 M sodium arsenite was rapidly added, and the reaction tubes were immersed in a boiling water bath for 3 min to end the reaction. Arsenate and arsenite were separated as described above. Partial Purification of Arsenate Reductase. Human liver was divided into 5 g pieces, minced with scissors, and homogenized with 10 mL of homogenization buffer in a Dounce homogenizer. Homogenization buffer contained 20 mM Tris-HCl (pH 8.0), 5 mM EDTA, 1 mM DTT, 0.2 mM PMSF, 100 mg/L ovomucoid, and 2.5 µM E-64. The homogenate was centrifuged at 12000g for 15 min at 4 °C. Lipids were removed from the supernatant by aspiration, and the supernatant was then centrifuged at 105000g for 90 min at 4 °C. Again lipids were removed from the supernatant by filtration through glass wool, and the resulting cytosol (designated fraction I) was stored at -70 °C. Cytosol (75 mL containing 4800 mg of protein) was diluted 1:1 with 10 mM Tris-HCl buffer (pH 7.35) and loaded onto a 2.5 cm × 15 cm DEAE-Sephacel column equilibrated with 30 mM Tris-HCl (pH 7.35) at 4 °C. The column was washed with 45 mL of 10 mM Tris-HCl buffer (pH 7.35) and then eluted with a gradient of 250 mL of 30 mM Tris-HCl (pH 7.35) and 250 mL of 30 mM Tris-HCl/0.5 M NaCl (pH 7.35). The flow rate was 6.1 cm h-1. Fractions (4.8 mL) were collected and immediately assayed for arsenate reductase activity and protein concentration. Fractions 25-43 were pooled and concentrated by ultrafiltration using a 30 kDa molecular mass cutoff membrane. The retentate (designated fraction II) was stored at -70 °C. The ultrafiltrate was also stored at -70 °C for use in
assaying fractions in the next purification step. This was done because no activtiy was detected in the retentate or ultrafiltrate; however, when the two were combined, activtiy was fully restored, suggesting a cofactor had been removed during ultrafiltration. Fraction II (3.5 mL containing 712 mg of protein) was thawed and loaded onto a 2.5 cm × 64 cm Sephacryl S-200 HR column equilibrated with 30 mM Tris-HCl/50 mM NaCl (pH 7.35), and the column was eluted with the same buffer at a flow rate of 4.9 cm h-1. Fractions (5 mL) were collected and immediately assayed for arsenate reductase activity and protein concentration. The ultrafiltrate (10 µL) from the previous step was added to the assay. Fractions 28-31 were pooled and concentrated by ultrafiltration. The retentate (designated fraction III) was stored at -70 °C. Determination of Molecular Mass by Size Exclusion Chromatography. A 2.5 cm × 56.4 cm Sephacryl S-200 HR column with a void volume (V0) determined with blue dextran 2000 was used for the molecular mass determination. Aldolase (158 kDa), bovine serum albumin (67 kDa), and ovalbumin (43 kDa) were used as molecular mass standards. Each standard (3 mL) or fraction II (3 mL) was run separately on the column at a flow rate of 6.1 cm h-1. Fractions (3 mL) were collected, and the chromatographic elution pattern of proteins was determined. The elution volume of the enzyme was determined using the standard enzyme assay. All standards and the enzyme were run on the column twice for reproducibility. Preparation of the Cofactor Solution. To have a reliable assay during later purification steps, a cofactor solution was prepared. This was done by making a liver cytosol preparation as described above but using 30 mM Tris-HCl (pH 7.35) as the homogenization buffer. The cytosol was placed in a boiling water bath for 10 min. The resulting solution was centrifuged for 1 h at 12000g, and the supernatant was filtered through a Centricon Plus filter (Amicon Inc.) with a 3 kDa membrane. The ultrafiltrate (designated cofactor preparation) was stored at -20 °C. Arsenate Reductase Specificity. The arsenate reductase assay was performed as described above using [14C]MMAV as the substrate to examine the specificity of the arsenate reductase. The assays were performed at pH 7.0 and 8.0. MMAV and MMAIII were separated on a Hamilton PRP-X100 anion exchange column using 30 mM sodium phosphate (pH 5.0) as the mobile phase at a flow rate of 1.0 mL min-1. A Beckman 171 radioisotope detector was used to quantify the methylated metabolites. Other Methods. The protein concentration was determined using the method of Bradford with catalase as the standard (20).
Results Partial Purification of Arsenate Reductase. The arsenate reductase specific activity of human liver cytosol was 0.9 unit/mg of protein (a unit represents the reduction of 1 nmol of arsenate per 30 min). After DEAESephacel and Sephacryl S-200 chromatography (Figures 1 and 2), there was a 24-fold purification (Table 1). Molecular Mass of Arsenate Reductase. V0 for the Sephacryl S-200 HR size exclusion column was 102 mL as determined with blue dextran 2000. Ve for arsenate reductase was 132 mL with a calculated Kav of 0.1829. The chromatography of standards and the enzyme was repeated, and the calculated molecular mass of the
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Chem. Res. Toxicol., Vol. 13, No. 1, 2000
Radabaugh and Aposhian
Figure 1. DEAE-Sephacel anion exchange chromatography of human liver arsenate reductase. Assay conditions are described in Experimental Procedures.
Figure 4. (A) Linearity of the arsenate reductase assay as a function of protein concentration. (B) Linearity of the arsenate reductase assay as a function of time. Fifty micrograms of protein from fraction III was used in each assay and corrected for nonenzymatic reduction by subtracting the amount of arsenite formed in assays containing 500 µmol of DTT but no protein (0). Reduction by the controls containing DTT but no protein is also shown (b). Each point represents the mean ( SE for three experiments.
Figure 2. Sephacryl S-200 HR size exclusion chromatography of human liver arsenate reductase. Assay conditions are described in Experimental Procedures.
Figure 3. Standard curve for molecular mass determination. The molecular mass of human liver arsenate reductase was 71.8 kDa. Assay conditions are described in Experimental Procedures.
human liver arsenate reductase based on the standard curve was 71.8 kDa (Figure 3). Linearity of the Arsenate Reductase Assay with Respect to Protein Concentration and Time. Using fraction III, it was determined that addition of 25-50 µg of protein to the reaction mixture resulted in a
corresponding increase in activity. Incubation of 50 µg of protein from fraction III showed increased arsenate reductase activity from 10 to 40 min. An incubation time of 30 min was adopted for the remainder of the experiments (Figure 4). A Heat Stable Cofactor Is Required. After DEAESephacel chromatography, peak fractions were pooled and concentrated by ultrafiltration. No activity in the retentate or ultrafiltrate could be detected. When the retentate and the ultrafiltrate were recombined, full activity was restored. The cofactor was in the ultrafiltrate. It was concluded a cofactor was required to assay the enzyme during later purification procedures. Therefore, a cofactor stock solution was prepared as described in Experimental Procedures. The filtrate was used as the stock solution of the required cofactor. This small heat stable cofactor is required for human liver arsenate reductase activity. Addition of ZnCl2, CuCl2, CaCl2, MnCl2, and MgCl2 at final concentrations of 10-3, 10-5, 10-6, or 10-7 M in the absence of the cofactor preparation resulted in activity that was less than 1% of the activity observed when 10 µL of 2-fold diluted cofactor solution was added (data not shown). When EDTA was present at concentrations up to 5 mM in assays containing the cofactor preparation, it did not inhibit the reductase activity, suggesting the cofactor is not an EDTA chelatable divalent cation. A Thiol Is Required for Arsenate Reductase. In addition, a thiol (e.g., DTT) was required for activity. The cofactor preparation itself had no arsenate reducing activity. The enzyme without cofactor was completely inactive. Significant activity is observed only in the
Human Liver Arsenate Reductase
Chem. Res. Toxicol., Vol. 13, No. 1, 2000 29
Table 2. Requirements of Human Liver Arsenate Reductase reaction
units
completea without enzyme without 0.5 mM DTT without cofactor preparation
1.2
