JOURNAL
OF
Vol. 173, No. 6
BACTERIOLOGY, Mar. 1991, p. 1938-1943
0021-9193/91/061938-06$02.00/0 Copyright C) 1991, American Society for Microbiology
Purification and Properties of an Organophosphorus Acid Anhydrase from a Halophilic Bacterial Isolate JOSEPH J. DEFRANK* AND TU-CHEN CHENG U.S. Army Chemical Research, Development & Engineering Center, Biotechnology Division, Research Directorate, Aberdeen Proving Ground, Maryland 21010-5423 Received 18 October 1990/Accepted 14 January 1991
A moderately halophilic bacterial isolate has been found to possess high levels of enzymatic activity against several highly toxic organophosphorus compounds. The predominant enzyme, designated organophosphorus acid anhydrase 2, has been purified 1,000-fold to homogeneity and characterized. The enzyme is a single polypeptide with a molecular weight of 60,000. With diisopropylfluorophosphate as a substrate, the enzyme has optimum activity at pH 8.5 and 50°C, and it is stimulated by manganese and cobalt. the springs are a relatively constant temperature of 24°C, a pH of 6.0, and a salt content of approximately 24% (14). Cultures were grown in a medium consisting of the following (grams per liter): NaCl, 50; MgSO2 7H20, 10; Proteose Peptone (Difco), 10; yeast extract, 6; and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 2.5 (pH 6.8). Inoculated flasks (4 or 6 liter) containing 1 to 1.5 liters of medium were incubated at 30 to 37°C, on a rotary shaker at 240 rpm, for 18 to 24 h. Cells were harvested by centrifugation (7,500 x g) at 20°C (to prevent precipitation of an unidentified saltlike material observed at lower temperatures) and stored at -20°C. Enzyme assays. OPA anhydrase activity was routinely assayed by monitoring fluoride release from DFP by an ion-specific electrode as has been described numerous times in the literature (6, 7, 11). Unless otherwise stated the reaction medium contained 500 mM NaCl, 50 mM Bis-Tris propane (pH 7.2), 3.0 mM DFP, 1.0 mM MnCl2 4H20, and 5 to 25 jil of enzyme sample in a total volume of 2.5 ml. The pH of 7.2 was selected to be the standard for all assays in order to be consistent with the numerous published reports on other OPA anhydrases (5-7, 10-13). Assays were run at 25°C in a temperature-controlled vessel with stirring. The enzyme sample was preincubated in the reaction medium for 1 min before the reaction was initiated by the addition of DFP (0.3 M in isopropanol). The reaction was monitored for 4 min, and the rate of fluoride release was corrected for spontaneous DFP hydrolysis under identical conditions. One unit of OPA anhydrase activity is defined as catalyzing the release of 1.0 ,umol of F- per min. Specific activity is expressed as units per milligram of protein. The hydrolysis of chromogenic substrates was conducted in a reaction mixture identical to that described above except that the substrate concentration was reduced to 5 to 100 puM. Activity was determined by monitoring the increase in absorbance at 405 nm (for p-nitrophenol), and units are expressed as 1.0 ,umol of p-nitrophenol released per min. The concentration of p-nitrophenol was determined from a standard curve with authentic material. Enzyme purification. All procedures were conducted at 4°C, and all centrifugations were at 46,000 x g for 30 min. Frozen or freshly harvested cells from 10 liters of culture were resuspended in 10BM buffer (10 mM Bis-Tris propane, 0.1 mM MnCl2 [pH 7.2]) at a ratio of 3 ml of buffer for each gram (wet weight) of cells. The cells were disrupted by passage through a French pressure cell (SLM Aminco) at
Organophosphorus acid (OPA) anhydrases are enzymes that are capable of catalytically hydrolyzing a wide variety of organophosphorus cholinesterase inhibitors, among them diisopropylfluorophosphate (DFP), the chemical warfare agents soman (0-1,2,2-trimethylpropyl methylphosphonofluoridate), sarin (O-isopropyl methylphosphonofluoridate), and tabun (ethyl N,N-dimethylphosphoramidocyanidate), and the pesticides parathion (diethyl p-nitrophenyl phosphorothioate) and paraoxon (diethyl p-nitrophenyl phosphate) (10, 12, 13). Enzymes such as these are of interest for their potential use in decontamination and demilitarization of these extremely toxic materials. In the past, these enzymes were known variously as DFPases, somanases, parathion hydrolases, or paraoxonases, depending on the assay substrate used. Sources of these enzymes range from bacteria and protozoans to higher mammals, including humans, and the number of enzymes found has greatly increased in recent years (10). The proliferation of both enzymes and enzyme names led to the adoption of the name OPA anhydrase during the First DFPase Workshop (Marine Biological Laboratory, Woods Hole, Mass., June 1987) to describe these related enzymes. It was planned that this name be used until the natural substrates and functions of these enzyme are identified. Preliminary studies of OPA anhydrases from various sources have demonstrated that these enzymes differ in substrate specificity, sensitivity to inhibitors, activation by metals, and molecular weight (10). Purification and characterization of these enzymes, such as the one described in this report, may assist in the determination of the true nature of their substrates, specificity, and molecular structure. The source of the enzyme to be discussed is the obligately halophilic bacterial isolate designated JD6.5, which was isolated from a warm salt spring. This isolate was found to possess high levels of DFP-hydrolyzing OPA anhydrase activity (3). In this report we describe the purification and characterization of OPA anhydrase 2 (OPAA-2), the predominant enzyme from JD6.5. MATERIALS AND METHODS
Organism and cultivation. Isolate JD6.5 was obtained from Grantsville Warm Springs, which is located approximately 30 miles (ca. 48 km) west of Salt Lake City, Utah, and just south of the Great Salt Lake. The primary characteristics of *
Corresponding author. 1938
VOL. 173, 1991
OPA ANHYDRASE FROM A HALOPHILE
0
E
E
0
0
M-
2
C'U a
4)-
o
0
c
0
0c
1939
_
_
._
o
L cU
0.z
0
4
O&0
,
.)
0 -
.4
0
1 00
200
300
Fraction No.
Fraction No.
FIG. 1. Separation of OPAA-1 and OPAA-2 by DEAE-Sephacel chromatography. Conditions are described in the text. Symbols: protein; --, NaCl; 0, OPA anhydrase activity.
16,000 lb/in2. Cellular debris was removed by centrifugation. The crude cell supernatant, which contained the OPA anhydrase activity, was treated with protamine sulfate to a final concentration of 0.4% in order to remove nucleic acids and associated proteins. After centrifugation, the supernatant was fractionated with solid (NH4)2SO4 to give the 30 to 65% (saturation) precipitate. The pellet was resuspended in a minimal volume of 10BM buffer and dialyzed against several changes of 20 volumes of the same buffer. The dialyzed sample was applied to a DEAE-Sephacel (Pharmacia) column (5 by 20 cm) previously equilibrated with 10BM buffer. The column was washed with 10BM to remove nonbinding materials. After the washing, the elution buffer was stepped to 200 mM NaCl in 10BM. The OPAA-2 activity was eluted with a 4-liter linear gradient of 200 to 600 mM NaCl (Fig. 1). Active fractions (193 to 212) were pooled, concentrated by precipitation at 65% (NH4)2SO4, and then centrifuged. The pellet was dissolved in 10 mM Bis-Tris propane-100 mM NaCl-5 mM KH2PO4 (pH 7.2). The solution was dialyzed overnight against 10 liters of this buffer. The enzyme solution was loaded onto a hydroxyapatite (HA-Ultrogel; IBF Biotechnics) column (2.6 by 14 cm) previously equilibrated with the Bis-Tris propane-NaClKH2PO4 (pH 7.2) buffer described above. (Manganese was not used during this chromatographic procedure to prevent precipitation of MnPO4.) After nonbinding protein was removed by washing, elution was carried out with a linear gradient of 5 to 150 mM KH2PO4 (Fig. 2). Enzyme fractions (52 to 58) were again pooled and concentrated by 65%
FIG. 2. Purification of OPAA-2 on HA-Ultrogel. Conditions are described in the text. Symbols: protein; --, phosphate; 0, OPA anhydrase activity. -,
(NH4)2SO4. After centrifugation, the pellet was redissolved in 10BM, supplemented with 10 mM NaCl (pH 7.2), and dialyzed against 6 liters of this buffer. The enzyme solution was further purified on an HPLC (high-performance liquid chromatography)-GTi system (LKB) using two GF-250 columns (0.94 by 25 cm; DuPont) in series and run with the 10BM-10 mM NaCl (pH 7.2) buffer at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were collected. The pooled enzyme fractions were concentrated with a Centricon-30 concentrator (Amicon) and loaded onto a 7% polyacrylamide gel. Electrophoresis was performed according to Laemmli (9) but without sodium dodecyl sulfate (SDS) and dithiothreitol (DTT). Immediately after the electrophoresis, the bands containing enzyme activity were cut out of the gel and eluted into the same electrophoresis buffer with an Extraphor electrophoretic concentrator (LKB). The results of these purification procedures are summarized in Table 1. SDS-PAGE and protein determination. SDS-polyacrylamide gel electrophoresis (PAGE) was performed according to Laemmli (9). Protein samples were boiled in loading buffer, in the presence or absence or DTT, prior to running. The protein bands were visualized with the Gelcode silver stain kit (Pierce). The Coomassie protein assay reagent (Pierce) was used for determination of protein concentrations, with bovine serum albumin as the standard. Preparation of polyclonal and monoclonal antibodies. The polyclonal antiserum to OPAA-2 was prepared by immunizing a rat (Sprague-Dawley) with the HPLC-purified enzyme
TABLE 1. Summary of purification of OPAA-2 from JD6.5 Purification step
Crude extract Protamine sulfate Ammonium sulfate DEAE-Sephacel HA-Ultrogel HPLC-GF 250 PAGE
Vol
Protein
(ml)
(mg) 3,005.0 2,938.0 1,167.0
306.0 328.0 145.0 19.0 1.3 2.0 4.0
19.4 0.8 0.4 0.1
Activity (U) 801.0 853.0 992.0 570.0 205.0 143.0 26.8
Sp act (U/mg) 0.267 0.290 0.850 29.38 259.49 357.50 268.00
Purification
(fold) 1.1 3.2 110.0 971.9
1,337.1 1,003.7
Yield (%)
100.0 106.5 123.8 71.2 25.6 17.9 3.3
J. BACTERIOL.
DEFRANK AND CHENG
1940
A
MW
MW
Af -
B
B
MW
200,000I-~
200,000 200,00097,000
97,000
5w
97,000-
-
68,000I-m
:_:a
-
68, 000_
3X
-
--
m
4
3
43,000
43,000-
43,000
1
25,7006
7
8
1
2
3
FIG. 3. (A) SDS-PAGE of fractions from JD6.5 OPAA-2 purification. Lanes 1 to 8 contain crude extract, protamine sulfate supernatant, ammonium sulfate pool, pooled DEAE-Sephacel fractions, pooled HA-Ultrogel fractions, HPLC fractions, preparative PAGE-purified OPAA-2, and molecular weight (MW) standards, respectively. (B) SDS-PAGE of purified OPAA-2 under reducing (lane 2) and nonreducing (lane 3) conditions. Lane 1 contains molecular weight standards.
fraction. The rat was first given a footpad injection of 100 ,ug of enzyme in complete adjuvant (Bacto). Three weeks later it received a subcutaneous booster of 20 ,ug in incomplete adjuvant (Bacto). One week later, a prefusion dose of 5 ,ug in sterile saline was administered intravenously. The withdrawal of polyclonal serum and the fusion procedure were started 3 days after the final injection. Serum was obtained by tail bleeding. Spleen cells from the immunized rat were removed and fused with mouse myeloma cell line SP2/0Ag14 (8). The hybridoma cell clone was detected by enzymelinked immunosorbent assay, using microtiter plates coated with a crude enzyme preparation. Biotinylated rabbit anti-rat immunoglobulin G conjugated to horseradish peroxidase (rat Extravidin staining kit; Sigma) was used for the detection of antibodies against OPAA-2. For Western immunoblotting analysis, the same detection kit was used as in the immunodetection assay. RESULTS
Organism. Isolate JD6.5 is a gram-negative, aerobic, short rod and an obligate halophile that requires at least 2% NaCl for growth (2). Fatty acid analysis (Microbial ID, Newark, Del.) has tentatively identified JD6.5 as a species of Alteromonas, but not the haloplanktis or putrefaciens species that were in the data base. Purification of OPAA-2. The enzyme preparations at different stages in the purification process (Table 1) were analyzed by SDS-PAGE (Fig. 3A). The purification protocol described above yielded an enzyme preparation that appeared to be homogeneous, as judged by a single band with a molecular weight of approximately 60,000. In a second gel analysis (Fig. 3B), no difference was observed in the electrophoretic mobility of purified OPAA-2 under reducing or
2
3
1
2
4
FIG. 4. Representation of Western blot with monoclonal (A) and polyclonal (B) antibodies. For both blots, lanes 1 to 4 represent crude extract, pooled DEAE-Sephacel fractions (OPAA-1), pooled DEAE-Sephacel fractions (OPAA-2), and purified OPAA-2, respectively. MW, Molecular weight standards.
nonreducing conditions. The results strongly suggest that the final purified enzyme is a single polypeptide. The purified OPAA-2, in addition to the pooled fractions 1 and 2 from the DEAE-Sephacel step and crude extract, was also analyzed by Western blotting after SDS-PAGE. The blots were analyzed with either monoclonal antibody 6 (Fig. 4A) or polyclonal antiserum (Fig. 4B). A single band of purified OPAA-2 was detected on both blots (lanes 4). The monoclonal antibody was also shown to react with two protein bands (molecular weights of 78,000 and 74,000) in both the crude extract and pooled fraction 1 (Fig. 4A, lanes 1 and 2). These proteins presumably make up the OPAA-1 peak observed in Fig. 1. In the blot reacted with antiserum, these two bands and a third band with an estimated molecular weight of 53,000 were also detected (Fig. 4B, lanes 1 and 2). The results suggest that these three protein bands are separated from OPAA-2 during the DEAE-Sephacel chromatography. The fact that both the monoclonal antibody and antiserum react more strongly to OPAA-1 than OPAA-2 suggests two possible explanations. OPAA-1 may be the precursor(s) for OPAA-2 and produced at high concentrations but with low specific activity against DFP. Alternatively, these proteins may all be descended from a common ancestral protein and still retain antigenic similarity while possibly differing significantly in specificity. To characterize the antiserum in terms of biological activity, the enzyme activity of purified OPAA-2 was determined after reaction with dilutions of the antiserum. In these preliminary experiments, the purified enzyme (250 ng) was incubated with dilutions of the antiserum in 25 ,ul of 10BM buffer at room temperature for 5 min prior to being assayed with DFP under standard conditions. At a 250-fold dilution of antiserum, a 50% inhibition of enzyme activity was observed (Table 2). Substrate specificity. The specific activities of OPAA-2 against a variety of substrates is summarized are Table 3. Because of the greater sensitivity of the spectrophotometric assay, lower concentrations of chromogenic substrates were used. Of the substrates tested, the highest activity was with DFP. The enzyme also exhibited activity against two chromogenic compounds, p-nitrophenylmethyl(phenyl)phosphinate (NPMPP) and p-nitrophenylethyl(phenyl)phosphinate
VOL. 173, 1991
OPA ANHYDRASE FROM A HALOPHILE
1941
TABLE 2. Inhibition of OPAA-2 by antiserum % Activity Normal rat serum + OPAA-2
Serum dilution
Antiserum + OPAA-2
1:100 1:250 1:500 1:1,000 1:2,000 Control, no serum Control, no enzyme
44.4 49.3 60.6 71.3 83.0
103.0 100.0 98.2
1.0b
1.0b
ND
b
60 40-
100lo.
20-
(NPEPP). Paraoxon was hydrolyzed by the purified enzyme at about 3 to 4% the rate of DFP. These results are still preliminary, and additional parameters such as Ki,m Vmax, and pH effect for each of these substrates remain to be
determined. In addition to these compounds, a variety of potential substrates for esterases, phosphatases, phosphdiesterases, phosphotriesterases, and phospholipases were examined as potential substrates. These compounds showed little or no activity with OPAA-2. Mipafox (N,N'-diisopropyl phosphorodiamidofluoridate), which is only slightly, if at all, hydrolyzed by OPAA-2, exhibited a significant level of inhibition of the enzyme for DFP hydrolysis. Figure 5 shows the effect of incubation of OPAA-2 with Mipafox. In this assay, Mipafox was added to the reaction mixture during the preincubation period before the addition of DFP. At 3.0 mM Mipafox, the DFP hydrolysis was inhibited by greater than 90%. As has been demonstrated with OPA anhydrases from hog kidney and Escherichia coli, this inhibition is competitive and reversible (5). Dialysis of the inhibited enzyme overnight against 10BM buffer containing 100 mM NaCl resulted in complete restoration of activity against DFP (1). Effect of pH and temperature. With DFP as a substrate, the effect of pH on the activity of OPAA-2 was examined; in addition, the kinetic parameters (4) were determined at each pH value (Fig. 6). The apparent pH optimum for activity (Kcat) for OPAA-2 was found to be 8.5. The highest level of catalytic efficiency (KcatlKm) was observed at pH 6.8. This value reflects the pKa of the enzyme and the identity of
0 0.0
I
2.0
1.0
3.0
Mlpafox [mM] FIG. 5. Inhibition of OPAA-2 by preincubation with increasing concentrations of Mipafox. Conditions are described in the text.
ionizing groups in the active site or on the surface. From these observations, the presence of a histidine or a cysteine at its catalytic site is suggested (4). The effect of temperature on the reaction rate of the hydrolysis of DFP by OPAA-2 was examined. The initial reaction rate of the enzyme with 3.0 mM DFP, at pH 7.2, reached its maximum at 50°C in the presence of 1.0 mM MnCl2. The level of activity decreased at 55°C, which was the highest temperature tested in order to protect the fluoride electrode. The purified enzyme could be stored for months at -70°C in the presence of DTT. The addition of DTT not only improved stability but appeared to stimulate activity over
TABLE 3. Substrate specificities of OPAA-2a Substrate Substrate
DFP NPMPP NPEPP Paraoxon
Mipafox p-Nitrophenyl acetate p-Nitrophenyl phosphate Bis(p-nitrophenyl) phosphate Tris(p-nitrophenyl) phosphate p-Nitrophenyl(phenyl) phosphonate p-Nitrophenyl phosphorylcholine
~Concn
% Activity +Manganese
((mM) -Manganese 3.0 0.1 0.1 0.1 1.0 0.1 0.1 0.1 0.1 0.1 0.1
71.3 35.1 28.3 2.8
OF
Vol. 173, No. 6
BACTERIOLOGY, Mar. 1991, p. 1938-1943
0021-9193/91/061938-06$02.00/0 Copyright C) 1991, American Society for Microbiology
Purification and Properties of an Organophosphorus Acid Anhydrase from a Halophilic Bacterial Isolate JOSEPH J. DEFRANK* AND TU-CHEN CHENG U.S. Army Chemical Research, Development & Engineering Center, Biotechnology Division, Research Directorate, Aberdeen Proving Ground, Maryland 21010-5423 Received 18 October 1990/Accepted 14 January 1991
A moderately halophilic bacterial isolate has been found to possess high levels of enzymatic activity against several highly toxic organophosphorus compounds. The predominant enzyme, designated organophosphorus acid anhydrase 2, has been purified 1,000-fold to homogeneity and characterized. The enzyme is a single polypeptide with a molecular weight of 60,000. With diisopropylfluorophosphate as a substrate, the enzyme has optimum activity at pH 8.5 and 50°C, and it is stimulated by manganese and cobalt. the springs are a relatively constant temperature of 24°C, a pH of 6.0, and a salt content of approximately 24% (14). Cultures were grown in a medium consisting of the following (grams per liter): NaCl, 50; MgSO2 7H20, 10; Proteose Peptone (Difco), 10; yeast extract, 6; and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 2.5 (pH 6.8). Inoculated flasks (4 or 6 liter) containing 1 to 1.5 liters of medium were incubated at 30 to 37°C, on a rotary shaker at 240 rpm, for 18 to 24 h. Cells were harvested by centrifugation (7,500 x g) at 20°C (to prevent precipitation of an unidentified saltlike material observed at lower temperatures) and stored at -20°C. Enzyme assays. OPA anhydrase activity was routinely assayed by monitoring fluoride release from DFP by an ion-specific electrode as has been described numerous times in the literature (6, 7, 11). Unless otherwise stated the reaction medium contained 500 mM NaCl, 50 mM Bis-Tris propane (pH 7.2), 3.0 mM DFP, 1.0 mM MnCl2 4H20, and 5 to 25 jil of enzyme sample in a total volume of 2.5 ml. The pH of 7.2 was selected to be the standard for all assays in order to be consistent with the numerous published reports on other OPA anhydrases (5-7, 10-13). Assays were run at 25°C in a temperature-controlled vessel with stirring. The enzyme sample was preincubated in the reaction medium for 1 min before the reaction was initiated by the addition of DFP (0.3 M in isopropanol). The reaction was monitored for 4 min, and the rate of fluoride release was corrected for spontaneous DFP hydrolysis under identical conditions. One unit of OPA anhydrase activity is defined as catalyzing the release of 1.0 ,umol of F- per min. Specific activity is expressed as units per milligram of protein. The hydrolysis of chromogenic substrates was conducted in a reaction mixture identical to that described above except that the substrate concentration was reduced to 5 to 100 puM. Activity was determined by monitoring the increase in absorbance at 405 nm (for p-nitrophenol), and units are expressed as 1.0 ,umol of p-nitrophenol released per min. The concentration of p-nitrophenol was determined from a standard curve with authentic material. Enzyme purification. All procedures were conducted at 4°C, and all centrifugations were at 46,000 x g for 30 min. Frozen or freshly harvested cells from 10 liters of culture were resuspended in 10BM buffer (10 mM Bis-Tris propane, 0.1 mM MnCl2 [pH 7.2]) at a ratio of 3 ml of buffer for each gram (wet weight) of cells. The cells were disrupted by passage through a French pressure cell (SLM Aminco) at
Organophosphorus acid (OPA) anhydrases are enzymes that are capable of catalytically hydrolyzing a wide variety of organophosphorus cholinesterase inhibitors, among them diisopropylfluorophosphate (DFP), the chemical warfare agents soman (0-1,2,2-trimethylpropyl methylphosphonofluoridate), sarin (O-isopropyl methylphosphonofluoridate), and tabun (ethyl N,N-dimethylphosphoramidocyanidate), and the pesticides parathion (diethyl p-nitrophenyl phosphorothioate) and paraoxon (diethyl p-nitrophenyl phosphate) (10, 12, 13). Enzymes such as these are of interest for their potential use in decontamination and demilitarization of these extremely toxic materials. In the past, these enzymes were known variously as DFPases, somanases, parathion hydrolases, or paraoxonases, depending on the assay substrate used. Sources of these enzymes range from bacteria and protozoans to higher mammals, including humans, and the number of enzymes found has greatly increased in recent years (10). The proliferation of both enzymes and enzyme names led to the adoption of the name OPA anhydrase during the First DFPase Workshop (Marine Biological Laboratory, Woods Hole, Mass., June 1987) to describe these related enzymes. It was planned that this name be used until the natural substrates and functions of these enzyme are identified. Preliminary studies of OPA anhydrases from various sources have demonstrated that these enzymes differ in substrate specificity, sensitivity to inhibitors, activation by metals, and molecular weight (10). Purification and characterization of these enzymes, such as the one described in this report, may assist in the determination of the true nature of their substrates, specificity, and molecular structure. The source of the enzyme to be discussed is the obligately halophilic bacterial isolate designated JD6.5, which was isolated from a warm salt spring. This isolate was found to possess high levels of DFP-hydrolyzing OPA anhydrase activity (3). In this report we describe the purification and characterization of OPA anhydrase 2 (OPAA-2), the predominant enzyme from JD6.5. MATERIALS AND METHODS
Organism and cultivation. Isolate JD6.5 was obtained from Grantsville Warm Springs, which is located approximately 30 miles (ca. 48 km) west of Salt Lake City, Utah, and just south of the Great Salt Lake. The primary characteristics of *
Corresponding author. 1938
VOL. 173, 1991
OPA ANHYDRASE FROM A HALOPHILE
0
E
E
0
0
M-
2
C'U a
4)-
o
0
c
0
0c
1939
_
_
._
o
L cU
0.z
0
4
O&0
,
.)
0 -
.4
0
1 00
200
300
Fraction No.
Fraction No.
FIG. 1. Separation of OPAA-1 and OPAA-2 by DEAE-Sephacel chromatography. Conditions are described in the text. Symbols: protein; --, NaCl; 0, OPA anhydrase activity.
16,000 lb/in2. Cellular debris was removed by centrifugation. The crude cell supernatant, which contained the OPA anhydrase activity, was treated with protamine sulfate to a final concentration of 0.4% in order to remove nucleic acids and associated proteins. After centrifugation, the supernatant was fractionated with solid (NH4)2SO4 to give the 30 to 65% (saturation) precipitate. The pellet was resuspended in a minimal volume of 10BM buffer and dialyzed against several changes of 20 volumes of the same buffer. The dialyzed sample was applied to a DEAE-Sephacel (Pharmacia) column (5 by 20 cm) previously equilibrated with 10BM buffer. The column was washed with 10BM to remove nonbinding materials. After the washing, the elution buffer was stepped to 200 mM NaCl in 10BM. The OPAA-2 activity was eluted with a 4-liter linear gradient of 200 to 600 mM NaCl (Fig. 1). Active fractions (193 to 212) were pooled, concentrated by precipitation at 65% (NH4)2SO4, and then centrifuged. The pellet was dissolved in 10 mM Bis-Tris propane-100 mM NaCl-5 mM KH2PO4 (pH 7.2). The solution was dialyzed overnight against 10 liters of this buffer. The enzyme solution was loaded onto a hydroxyapatite (HA-Ultrogel; IBF Biotechnics) column (2.6 by 14 cm) previously equilibrated with the Bis-Tris propane-NaClKH2PO4 (pH 7.2) buffer described above. (Manganese was not used during this chromatographic procedure to prevent precipitation of MnPO4.) After nonbinding protein was removed by washing, elution was carried out with a linear gradient of 5 to 150 mM KH2PO4 (Fig. 2). Enzyme fractions (52 to 58) were again pooled and concentrated by 65%
FIG. 2. Purification of OPAA-2 on HA-Ultrogel. Conditions are described in the text. Symbols: protein; --, phosphate; 0, OPA anhydrase activity. -,
(NH4)2SO4. After centrifugation, the pellet was redissolved in 10BM, supplemented with 10 mM NaCl (pH 7.2), and dialyzed against 6 liters of this buffer. The enzyme solution was further purified on an HPLC (high-performance liquid chromatography)-GTi system (LKB) using two GF-250 columns (0.94 by 25 cm; DuPont) in series and run with the 10BM-10 mM NaCl (pH 7.2) buffer at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were collected. The pooled enzyme fractions were concentrated with a Centricon-30 concentrator (Amicon) and loaded onto a 7% polyacrylamide gel. Electrophoresis was performed according to Laemmli (9) but without sodium dodecyl sulfate (SDS) and dithiothreitol (DTT). Immediately after the electrophoresis, the bands containing enzyme activity were cut out of the gel and eluted into the same electrophoresis buffer with an Extraphor electrophoretic concentrator (LKB). The results of these purification procedures are summarized in Table 1. SDS-PAGE and protein determination. SDS-polyacrylamide gel electrophoresis (PAGE) was performed according to Laemmli (9). Protein samples were boiled in loading buffer, in the presence or absence or DTT, prior to running. The protein bands were visualized with the Gelcode silver stain kit (Pierce). The Coomassie protein assay reagent (Pierce) was used for determination of protein concentrations, with bovine serum albumin as the standard. Preparation of polyclonal and monoclonal antibodies. The polyclonal antiserum to OPAA-2 was prepared by immunizing a rat (Sprague-Dawley) with the HPLC-purified enzyme
TABLE 1. Summary of purification of OPAA-2 from JD6.5 Purification step
Crude extract Protamine sulfate Ammonium sulfate DEAE-Sephacel HA-Ultrogel HPLC-GF 250 PAGE
Vol
Protein
(ml)
(mg) 3,005.0 2,938.0 1,167.0
306.0 328.0 145.0 19.0 1.3 2.0 4.0
19.4 0.8 0.4 0.1
Activity (U) 801.0 853.0 992.0 570.0 205.0 143.0 26.8
Sp act (U/mg) 0.267 0.290 0.850 29.38 259.49 357.50 268.00
Purification
(fold) 1.1 3.2 110.0 971.9
1,337.1 1,003.7
Yield (%)
100.0 106.5 123.8 71.2 25.6 17.9 3.3
J. BACTERIOL.
DEFRANK AND CHENG
1940
A
MW
MW
Af -
B
B
MW
200,000I-~
200,000 200,00097,000
97,000
5w
97,000-
-
68,000I-m
:_:a
-
68, 000_
3X
-
--
m
4
3
43,000
43,000-
43,000
1
25,7006
7
8
1
2
3
FIG. 3. (A) SDS-PAGE of fractions from JD6.5 OPAA-2 purification. Lanes 1 to 8 contain crude extract, protamine sulfate supernatant, ammonium sulfate pool, pooled DEAE-Sephacel fractions, pooled HA-Ultrogel fractions, HPLC fractions, preparative PAGE-purified OPAA-2, and molecular weight (MW) standards, respectively. (B) SDS-PAGE of purified OPAA-2 under reducing (lane 2) and nonreducing (lane 3) conditions. Lane 1 contains molecular weight standards.
fraction. The rat was first given a footpad injection of 100 ,ug of enzyme in complete adjuvant (Bacto). Three weeks later it received a subcutaneous booster of 20 ,ug in incomplete adjuvant (Bacto). One week later, a prefusion dose of 5 ,ug in sterile saline was administered intravenously. The withdrawal of polyclonal serum and the fusion procedure were started 3 days after the final injection. Serum was obtained by tail bleeding. Spleen cells from the immunized rat were removed and fused with mouse myeloma cell line SP2/0Ag14 (8). The hybridoma cell clone was detected by enzymelinked immunosorbent assay, using microtiter plates coated with a crude enzyme preparation. Biotinylated rabbit anti-rat immunoglobulin G conjugated to horseradish peroxidase (rat Extravidin staining kit; Sigma) was used for the detection of antibodies against OPAA-2. For Western immunoblotting analysis, the same detection kit was used as in the immunodetection assay. RESULTS
Organism. Isolate JD6.5 is a gram-negative, aerobic, short rod and an obligate halophile that requires at least 2% NaCl for growth (2). Fatty acid analysis (Microbial ID, Newark, Del.) has tentatively identified JD6.5 as a species of Alteromonas, but not the haloplanktis or putrefaciens species that were in the data base. Purification of OPAA-2. The enzyme preparations at different stages in the purification process (Table 1) were analyzed by SDS-PAGE (Fig. 3A). The purification protocol described above yielded an enzyme preparation that appeared to be homogeneous, as judged by a single band with a molecular weight of approximately 60,000. In a second gel analysis (Fig. 3B), no difference was observed in the electrophoretic mobility of purified OPAA-2 under reducing or
2
3
1
2
4
FIG. 4. Representation of Western blot with monoclonal (A) and polyclonal (B) antibodies. For both blots, lanes 1 to 4 represent crude extract, pooled DEAE-Sephacel fractions (OPAA-1), pooled DEAE-Sephacel fractions (OPAA-2), and purified OPAA-2, respectively. MW, Molecular weight standards.
nonreducing conditions. The results strongly suggest that the final purified enzyme is a single polypeptide. The purified OPAA-2, in addition to the pooled fractions 1 and 2 from the DEAE-Sephacel step and crude extract, was also analyzed by Western blotting after SDS-PAGE. The blots were analyzed with either monoclonal antibody 6 (Fig. 4A) or polyclonal antiserum (Fig. 4B). A single band of purified OPAA-2 was detected on both blots (lanes 4). The monoclonal antibody was also shown to react with two protein bands (molecular weights of 78,000 and 74,000) in both the crude extract and pooled fraction 1 (Fig. 4A, lanes 1 and 2). These proteins presumably make up the OPAA-1 peak observed in Fig. 1. In the blot reacted with antiserum, these two bands and a third band with an estimated molecular weight of 53,000 were also detected (Fig. 4B, lanes 1 and 2). The results suggest that these three protein bands are separated from OPAA-2 during the DEAE-Sephacel chromatography. The fact that both the monoclonal antibody and antiserum react more strongly to OPAA-1 than OPAA-2 suggests two possible explanations. OPAA-1 may be the precursor(s) for OPAA-2 and produced at high concentrations but with low specific activity against DFP. Alternatively, these proteins may all be descended from a common ancestral protein and still retain antigenic similarity while possibly differing significantly in specificity. To characterize the antiserum in terms of biological activity, the enzyme activity of purified OPAA-2 was determined after reaction with dilutions of the antiserum. In these preliminary experiments, the purified enzyme (250 ng) was incubated with dilutions of the antiserum in 25 ,ul of 10BM buffer at room temperature for 5 min prior to being assayed with DFP under standard conditions. At a 250-fold dilution of antiserum, a 50% inhibition of enzyme activity was observed (Table 2). Substrate specificity. The specific activities of OPAA-2 against a variety of substrates is summarized are Table 3. Because of the greater sensitivity of the spectrophotometric assay, lower concentrations of chromogenic substrates were used. Of the substrates tested, the highest activity was with DFP. The enzyme also exhibited activity against two chromogenic compounds, p-nitrophenylmethyl(phenyl)phosphinate (NPMPP) and p-nitrophenylethyl(phenyl)phosphinate
VOL. 173, 1991
OPA ANHYDRASE FROM A HALOPHILE
1941
TABLE 2. Inhibition of OPAA-2 by antiserum % Activity Normal rat serum + OPAA-2
Serum dilution
Antiserum + OPAA-2
1:100 1:250 1:500 1:1,000 1:2,000 Control, no serum Control, no enzyme
44.4 49.3 60.6 71.3 83.0
103.0 100.0 98.2
1.0b
1.0b
ND
b
60 40-
100lo.
20-
(NPEPP). Paraoxon was hydrolyzed by the purified enzyme at about 3 to 4% the rate of DFP. These results are still preliminary, and additional parameters such as Ki,m Vmax, and pH effect for each of these substrates remain to be
determined. In addition to these compounds, a variety of potential substrates for esterases, phosphatases, phosphdiesterases, phosphotriesterases, and phospholipases were examined as potential substrates. These compounds showed little or no activity with OPAA-2. Mipafox (N,N'-diisopropyl phosphorodiamidofluoridate), which is only slightly, if at all, hydrolyzed by OPAA-2, exhibited a significant level of inhibition of the enzyme for DFP hydrolysis. Figure 5 shows the effect of incubation of OPAA-2 with Mipafox. In this assay, Mipafox was added to the reaction mixture during the preincubation period before the addition of DFP. At 3.0 mM Mipafox, the DFP hydrolysis was inhibited by greater than 90%. As has been demonstrated with OPA anhydrases from hog kidney and Escherichia coli, this inhibition is competitive and reversible (5). Dialysis of the inhibited enzyme overnight against 10BM buffer containing 100 mM NaCl resulted in complete restoration of activity against DFP (1). Effect of pH and temperature. With DFP as a substrate, the effect of pH on the activity of OPAA-2 was examined; in addition, the kinetic parameters (4) were determined at each pH value (Fig. 6). The apparent pH optimum for activity (Kcat) for OPAA-2 was found to be 8.5. The highest level of catalytic efficiency (KcatlKm) was observed at pH 6.8. This value reflects the pKa of the enzyme and the identity of
0 0.0
I
2.0
1.0
3.0
Mlpafox [mM] FIG. 5. Inhibition of OPAA-2 by preincubation with increasing concentrations of Mipafox. Conditions are described in the text.
ionizing groups in the active site or on the surface. From these observations, the presence of a histidine or a cysteine at its catalytic site is suggested (4). The effect of temperature on the reaction rate of the hydrolysis of DFP by OPAA-2 was examined. The initial reaction rate of the enzyme with 3.0 mM DFP, at pH 7.2, reached its maximum at 50°C in the presence of 1.0 mM MnCl2. The level of activity decreased at 55°C, which was the highest temperature tested in order to protect the fluoride electrode. The purified enzyme could be stored for months at -70°C in the presence of DTT. The addition of DTT not only improved stability but appeared to stimulate activity over
TABLE 3. Substrate specificities of OPAA-2a Substrate Substrate
DFP NPMPP NPEPP Paraoxon
Mipafox p-Nitrophenyl acetate p-Nitrophenyl phosphate Bis(p-nitrophenyl) phosphate Tris(p-nitrophenyl) phosphate p-Nitrophenyl(phenyl) phosphonate p-Nitrophenyl phosphorylcholine
~Concn
% Activity +Manganese
((mM) -Manganese 3.0 0.1 0.1 0.1 1.0 0.1 0.1 0.1 0.1 0.1 0.1
71.3 35.1 28.3 2.8
