ANALYTICAL
BIOCHEMISTRY
83, 609-614 (1977)
An lmmunochemical Method for Determining Thioredoxin Reductase in Rat Tissues CHIN-CHUN CHEN AND E. COLLEEN MOORE Department of Biochemistry, M. D. Anderson Hospitai
The University of and Tumor Institute,
Texas System Cancer Center, Houston,
Texas
77030
ReceivedApril 25, 1977; accepted August 4, 1977 The immunochemical method, microcomplement fixation, was used in the assay of thioredoxin reductase. The method is more sensitive and specific than previously used methods. The enzyme in different tissues of rats was measured.
Thioredoxin reductase (EC 1.6.4.5 .) is an NADPH-linked flavoprotein which reduces thioredoxin, a small protein which in turn serves as the direct substrate of ribonucleotide reductase. Thioredoxin reductase has been reported in Escherichia coli (I), Lactobacillus leichmannii (2), yeast (3), mammalian livers (4,5), and tumor cells (6). The assay of the mammalian enzyme has been inconvenient, primarily because of the difficulty of obtaining and storing active thioredoxin. So far three methods have been used, and each had disadvantages. We wish to present here a highly sensitive and specific immunochemical method for the determination of thioredoxin reductase. It is especially useful with unpurified samples that frequently contain substances which interfere with the measurement of enzyme activity. The new method has been used to determine the level of thioredoxin reductase in several rat tissues. MATERIALS
AND METHODS
Materials. Sterile sheep red blood cells were obtained from Colorado Serum Company Laboratories. Complement extracted from guinea pig serum was obtained from Miles Laboratories and hemolysin for complement fixation from Flow Laboratories. Tissue sample preparation. Fresh or frozen rat tissues were washed with saline several times. The connective tissue was removed by forcing through a screen. The tissues then were homogenized with a Teflon-glass homogenizer in 3 vol of 1 mM sodium bisulfite solution. The homogenate was centrifuged at 100,OOOgfor an hour. The supernatant was used for the assay. 609 Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN WO3-2697
610
CHEN AND MOORE
Preparation of a~t~bo~~~ The pu~fication of thioredoxin reductase from tumor cells has been reported (7). The purified thioredoxin reductase, which contained only one protein band at 58,000 daltons on SDS-polyacrylamide gel electrophoresis, was used to immunize a New Zealand rabbit. Four injections of 100 pg, in Freund’s complete adjuvant, were given subcutaneously on the back at intervals of 7,28, and 39 days, and serum was collected 10 days after the last injection. The antibody was isolated by the following steps: (a) precipitating the antiserum twice with 40% ammonium sulfate at 5°C; (b) dialyzing against 0.0175~ phosphate buffer at pH 6.3 overnight; and (c) passing through a prepared DEAE column as described (8). The antibody obtained from the DEAE column was lyophilized, stored at -?O”C, and redissolved in distilled water when needed. The concentration of protein was determined by the method of Lowry et al. (9). ~~~~~~~~~~j~~. Immuno double diffusion tests were performed with 1% agar disks and 0.02 M Tris buffer at pH 7.5. Ten microliters of a concentrated solution of antigen and 25 ~1 of antiserum were placed in wells. Diffusion was performed at room temperature for 24 hr. The precipitins were photographed. Immunoelectrophoresis. The gels for immunoelectrophoresis contained 1.75% agarose with 0.04 M barbital buffer, pH 8.2, and 0.05% sodium azide. Two samples of enzyme were electrophoresed in a 2.5 x 7.5cm gel at 8 mA at 5°C for about 1 hr; 150 ~1 of the antibody was put into the trough, which was cut after electrophoresis, and was allowed to diffuse as described above. Complement fixation. Complement fixation was performed by the method of Levine (10). The total volume was reduced to 0.8 ml; I:400 hemolysin and 1600 complement were used. Samples to be assayed were diluted appropriately with diluent. About 0.1 to 10 fig of crude protein or 1 to 30 ng of purified enzyme was incubated with 22 pg of antibody and complement in 0.6 ml at 5°C overnight. To the mixture, 0.2 ml of 0.25% sensitized cells was added. The absorbance was read at 413 nm; at complete hemolysis it was usually in the range of 0.8 to 1.0. Bzzyme activity. Thioredoxin reductase activity was measured by coupling to ribonucleotide reductase from E. coli, as described [method 1 in Ref. (7)]. In one experiment with pure enzyme, the coupled reduction of glutathione by reduced thioredoxin was used [see Ref. (5) for a similar procedure]. RESULTS
With either the purified thioredoxin reductase or the crude supernatant from the Novikoff tumor homogenate, only a single precipitin band was detected by immuno double diffusion. These two samples also showed only a single arc of precipitin at the same position in immunoelectro-
THIOREDOXIN
REDUCTASE
611
ASSAY
phoresis, which indicated that the enzyme was the only antigen to the antibody. Using the antibody to detect thioredoxin reductase in different tissues, we found that homogenates of liver, thymus, spleen, heart, lung, kidney, and brain of rats formed the precipitin band with the antibody. However, serum and erythrocytes did not contain this antigen. To measure the enzyme quantitatively, a standard complement fixation curve of the purified enzyme was established, as shown in Fig. 1. The amount of the enzyme giving 50% fixation of complement in the presence of 22 pg of antibody was 6 ng. Each assay included a standard curve with known amounts of enzyme. The control for 100% lysis was the solution without antibody, and that for 0% lysis was the solution containing only diluent and the red blood cells. In order to compare the results of this method with those of enzyme activity measurements, a series of samples obtained during purification of the enzyme were measured by both the immunochemical method and by the most specific method for enzyme activity, a coupled reaction with ribonucleotide reductase, thioredoxin, [a32P]CDP and NADPH. The results are shown in Table 1. The last column is the ratio of the enzyme activity measured by the CDP method to the measured antigen. The ratio was almost constant throughout the purification. The method was used to determine enzyme (antigen) in several rat tissues. The amounts of the enzyme found are shown in Table 2. DISCUSSION
Thioredoxin reductase activity has previously been measured by the three following methods. The first method (CDP method) is the
100 I
80 20 B G” 60. E $
40.
if ”
20.
5
IO Antigen
15
I,
30
(ng)
FIG. 1. Standard curve for complement fixation by thioredoxin tube contained 22 pg of antibody. Homogeneous purified thioredoxin as shown on the abscissa. The procedure is described in Materials
reductase. Each assay reductase was added and Methods.
612
CHENANDMOORE TABLE COMPARISON
OF ENZYME
ACTIVITY
Immunochemical
method
Enzyme concentration (dml)
Step Ib II III IV V VI
97 169 166 18.5 139 363
WITH
1 ANTIGEN
Total enzyme (me)
Yield (%)
116 98 96 30.6 17.0 10.5
100 84 83 26 1.5 9
IN PURIFICATION
OF A BATCH
CDP method: Enzyme activity (units/mBa
Ratio of activity to antigen (units/fig)
2.83 4.91 2.96 5.97 3.36 11.73
0.029 0.029 0.018 0.032 0.024 0.032
(1One unit corresponds to reduction of 1 pmol of CDP in 30 min at 26”C, with a particular batch of ribonucleotide reductase and thioredoxin. Al1 samples were assayed at the same time. b Step I is the pH 5.2 supernatant; Step II is a DEAE-cellulose eluate; Step III is after heating to 55°C; Step IV is after ammonium sulfate precipitation; Step V is aRer Sephadex G-75 and a second DEAE-cellulose column; and Step VI is after adsorption and elution from calcium phosphate gel. The overall purification was from 0.29 units/mg of total protein in Step I to 26 units/mg.
stimulation of reduction of CDP determined in a system containing ribonucieotide reductase, thioredoxin, and NADPH. The measured activity varies with the amount of ribonucleotide reductase and thioredoxin used. The assay is tedious; its range is limited. The ribonucleotide reductase is unstable and the supply of these enzymes is limited. TABLE THIOREDOXIN
REDUCTASE
IN
DIFFERENT
Tissue Novikoff ascites tumor Liver Kidney
Thymus Spleen Lung Heart Brain
100,OOOg SUPERNATANTS RAT
Protein concentration (meiml) 16.6 24.5 16.2 9.2 18.0 14.3 7.4 7.4
2 FROM
TISSUES
Enzyme Owht)
Enzyme Protein (%I
0.102 0.068 0.029 0.010 0.012 0.013 0.0087 0.006
0.61 0.28 0.18 0.11 0.07 0.09 0.12 0.08
THIOREDOXIN
REDUCTASE
ASSAY
613
The second method is the coupled reduction of insulin [or glutathione, or for E. coli thioredoxin reductase, 5,5’-dithiobis-(2nitrobenzoic acid) (DTNB)] in the presence of thioredoxin and NADPH. The major disadvantage of this method is that the sample must be free of other enzymes generating (or consuming) sulfhydryl groups. A supply of thioredoxin is also required. The third method which has been used with mammalian thioredoxin reductase is the direct reduction of DTNB. Although this method is quick and simple, the reaction rate is not linear with the concentration of the enzyme so that it is not a quantitative method. None of these methods is really satisfactory with crude samples, due to either nonspecificity or interference by contaminants. Here we introduce an immunochemical method which avoids some of the previous problems. This method is both sensitive and specific. The specificity depends on the antibody, which requires that a highly purified enzyme be used as the antigen. The previous methods require 10 to 100 ng of enzyme whereas this method requires only 3 to 10 ng. It is thus up to 10 times more sensitive. Of course the new method does not directly measure the enzyme activity; it measures antigen. With 0.3 mg of purified enzyme one can obtain more than 300 mg of antibody which can be used in thousands of assays. In order to economize on pure enzyme a less pure sample can be used for the standard curve in complement fixation, providing it is first calibrated with the pure enzyme. In Table 2 we see that thioredoxin reductase is most abundant in tumor and liver. The thioredoxin reductase prepared from liver has an electrophoretic mobility slightly different from that of the tumor enzyme (Chen et al., paper in preparation). The ratio of enzyme activity to complement-fixing antigen, however, differs by less than 20% between the two tissues. When a sample of enzyme was modified by reduction with 1% 2-mercaptoethanol or by carboxymethylation with iodoacetate, the enzyme activity was lost but the ability to form precipitin remained. This suggests that the regions controlling the antigenicity are more stable than the region of the enzyme active site. The enzyme activity, as measured by coupled reduction of glutathione, decreased to 35% in the presence of excess antibody. The low activity/antigen ratio in Step III of Table 1 may be due to denaturation of some of the enzyme without destruction of its antigenicity, with the subsequent puritication step removing the denatured enzyme. ACKNOWLEDGMENTS We wish to thank Dr. J. Y. Wu at Baylor Coliege of Medicine for the gift of some reagents as well as for some valuable discussions. This work was supported by grants to E. C. Moore from the National Cancer Institute (CA 04464) and the Robert A. Welch Foundation (G-455).
614
CHEN AND MOORE
REFERENCES 1. 2. 3. 4. 5.
Moore, E. C., Reichard, P., and Thelander, L. (1964) 3. Biol. Ckem. 239, 344.5-3452. Orr, M. D., and Vitols, E. (1966) Biockem. Biopkys. Res. Commun. 25, 109-115. Porque, P. G., Baldesten, A., and Reichard, P. (1970)J. Biol. Ckem. 245, 2363-2370. Larsson, A. (1973) Eur. J. Biockem. 35, 346-349. Engstriim, N. E., Holmgren, A., Larsson, A., and Siiderhall, S. (1974)J. Biol. Ckem. 249,205-210.
6. Moore, E. C. (1967) Biockem. Biopkys. Res. Commun. 29, 264-268. 7. Chen, C. C., McCall, B. L. B., and Moore, E. C. (1977) Prep. Biockem. 7, 165-177. 8. Levy, H. B., and Sober, H. A. (1960) Proc. Sot. Exp. Biol. Med. 103, 250-252. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)J. Bioi. Ckem. 193,265-27X
10. Levine, L. (1967) in Handbook of Experimental 707-719, Blackwelf Scientific, Oxford.
immunology
(Weir, D. M., ed.), pp.
BIOCHEMISTRY
83, 609-614 (1977)
An lmmunochemical Method for Determining Thioredoxin Reductase in Rat Tissues CHIN-CHUN CHEN AND E. COLLEEN MOORE Department of Biochemistry, M. D. Anderson Hospitai
The University of and Tumor Institute,
Texas System Cancer Center, Houston,
Texas
77030
ReceivedApril 25, 1977; accepted August 4, 1977 The immunochemical method, microcomplement fixation, was used in the assay of thioredoxin reductase. The method is more sensitive and specific than previously used methods. The enzyme in different tissues of rats was measured.
Thioredoxin reductase (EC 1.6.4.5 .) is an NADPH-linked flavoprotein which reduces thioredoxin, a small protein which in turn serves as the direct substrate of ribonucleotide reductase. Thioredoxin reductase has been reported in Escherichia coli (I), Lactobacillus leichmannii (2), yeast (3), mammalian livers (4,5), and tumor cells (6). The assay of the mammalian enzyme has been inconvenient, primarily because of the difficulty of obtaining and storing active thioredoxin. So far three methods have been used, and each had disadvantages. We wish to present here a highly sensitive and specific immunochemical method for the determination of thioredoxin reductase. It is especially useful with unpurified samples that frequently contain substances which interfere with the measurement of enzyme activity. The new method has been used to determine the level of thioredoxin reductase in several rat tissues. MATERIALS
AND METHODS
Materials. Sterile sheep red blood cells were obtained from Colorado Serum Company Laboratories. Complement extracted from guinea pig serum was obtained from Miles Laboratories and hemolysin for complement fixation from Flow Laboratories. Tissue sample preparation. Fresh or frozen rat tissues were washed with saline several times. The connective tissue was removed by forcing through a screen. The tissues then were homogenized with a Teflon-glass homogenizer in 3 vol of 1 mM sodium bisulfite solution. The homogenate was centrifuged at 100,OOOgfor an hour. The supernatant was used for the assay. 609 Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN WO3-2697
610
CHEN AND MOORE
Preparation of a~t~bo~~~ The pu~fication of thioredoxin reductase from tumor cells has been reported (7). The purified thioredoxin reductase, which contained only one protein band at 58,000 daltons on SDS-polyacrylamide gel electrophoresis, was used to immunize a New Zealand rabbit. Four injections of 100 pg, in Freund’s complete adjuvant, were given subcutaneously on the back at intervals of 7,28, and 39 days, and serum was collected 10 days after the last injection. The antibody was isolated by the following steps: (a) precipitating the antiserum twice with 40% ammonium sulfate at 5°C; (b) dialyzing against 0.0175~ phosphate buffer at pH 6.3 overnight; and (c) passing through a prepared DEAE column as described (8). The antibody obtained from the DEAE column was lyophilized, stored at -?O”C, and redissolved in distilled water when needed. The concentration of protein was determined by the method of Lowry et al. (9). ~~~~~~~~~~j~~. Immuno double diffusion tests were performed with 1% agar disks and 0.02 M Tris buffer at pH 7.5. Ten microliters of a concentrated solution of antigen and 25 ~1 of antiserum were placed in wells. Diffusion was performed at room temperature for 24 hr. The precipitins were photographed. Immunoelectrophoresis. The gels for immunoelectrophoresis contained 1.75% agarose with 0.04 M barbital buffer, pH 8.2, and 0.05% sodium azide. Two samples of enzyme were electrophoresed in a 2.5 x 7.5cm gel at 8 mA at 5°C for about 1 hr; 150 ~1 of the antibody was put into the trough, which was cut after electrophoresis, and was allowed to diffuse as described above. Complement fixation. Complement fixation was performed by the method of Levine (10). The total volume was reduced to 0.8 ml; I:400 hemolysin and 1600 complement were used. Samples to be assayed were diluted appropriately with diluent. About 0.1 to 10 fig of crude protein or 1 to 30 ng of purified enzyme was incubated with 22 pg of antibody and complement in 0.6 ml at 5°C overnight. To the mixture, 0.2 ml of 0.25% sensitized cells was added. The absorbance was read at 413 nm; at complete hemolysis it was usually in the range of 0.8 to 1.0. Bzzyme activity. Thioredoxin reductase activity was measured by coupling to ribonucleotide reductase from E. coli, as described [method 1 in Ref. (7)]. In one experiment with pure enzyme, the coupled reduction of glutathione by reduced thioredoxin was used [see Ref. (5) for a similar procedure]. RESULTS
With either the purified thioredoxin reductase or the crude supernatant from the Novikoff tumor homogenate, only a single precipitin band was detected by immuno double diffusion. These two samples also showed only a single arc of precipitin at the same position in immunoelectro-
THIOREDOXIN
REDUCTASE
611
ASSAY
phoresis, which indicated that the enzyme was the only antigen to the antibody. Using the antibody to detect thioredoxin reductase in different tissues, we found that homogenates of liver, thymus, spleen, heart, lung, kidney, and brain of rats formed the precipitin band with the antibody. However, serum and erythrocytes did not contain this antigen. To measure the enzyme quantitatively, a standard complement fixation curve of the purified enzyme was established, as shown in Fig. 1. The amount of the enzyme giving 50% fixation of complement in the presence of 22 pg of antibody was 6 ng. Each assay included a standard curve with known amounts of enzyme. The control for 100% lysis was the solution without antibody, and that for 0% lysis was the solution containing only diluent and the red blood cells. In order to compare the results of this method with those of enzyme activity measurements, a series of samples obtained during purification of the enzyme were measured by both the immunochemical method and by the most specific method for enzyme activity, a coupled reaction with ribonucleotide reductase, thioredoxin, [a32P]CDP and NADPH. The results are shown in Table 1. The last column is the ratio of the enzyme activity measured by the CDP method to the measured antigen. The ratio was almost constant throughout the purification. The method was used to determine enzyme (antigen) in several rat tissues. The amounts of the enzyme found are shown in Table 2. DISCUSSION
Thioredoxin reductase activity has previously been measured by the three following methods. The first method (CDP method) is the
100 I
80 20 B G” 60. E $
40.
if ”
20.
5
IO Antigen
15
I,
30
(ng)
FIG. 1. Standard curve for complement fixation by thioredoxin tube contained 22 pg of antibody. Homogeneous purified thioredoxin as shown on the abscissa. The procedure is described in Materials
reductase. Each assay reductase was added and Methods.
612
CHENANDMOORE TABLE COMPARISON
OF ENZYME
ACTIVITY
Immunochemical
method
Enzyme concentration (dml)
Step Ib II III IV V VI
97 169 166 18.5 139 363
WITH
1 ANTIGEN
Total enzyme (me)
Yield (%)
116 98 96 30.6 17.0 10.5
100 84 83 26 1.5 9
IN PURIFICATION
OF A BATCH
CDP method: Enzyme activity (units/mBa
Ratio of activity to antigen (units/fig)
2.83 4.91 2.96 5.97 3.36 11.73
0.029 0.029 0.018 0.032 0.024 0.032
(1One unit corresponds to reduction of 1 pmol of CDP in 30 min at 26”C, with a particular batch of ribonucleotide reductase and thioredoxin. Al1 samples were assayed at the same time. b Step I is the pH 5.2 supernatant; Step II is a DEAE-cellulose eluate; Step III is after heating to 55°C; Step IV is after ammonium sulfate precipitation; Step V is aRer Sephadex G-75 and a second DEAE-cellulose column; and Step VI is after adsorption and elution from calcium phosphate gel. The overall purification was from 0.29 units/mg of total protein in Step I to 26 units/mg.
stimulation of reduction of CDP determined in a system containing ribonucieotide reductase, thioredoxin, and NADPH. The measured activity varies with the amount of ribonucleotide reductase and thioredoxin used. The assay is tedious; its range is limited. The ribonucleotide reductase is unstable and the supply of these enzymes is limited. TABLE THIOREDOXIN
REDUCTASE
IN
DIFFERENT
Tissue Novikoff ascites tumor Liver Kidney
Thymus Spleen Lung Heart Brain
100,OOOg SUPERNATANTS RAT
Protein concentration (meiml) 16.6 24.5 16.2 9.2 18.0 14.3 7.4 7.4
2 FROM
TISSUES
Enzyme Owht)
Enzyme Protein (%I
0.102 0.068 0.029 0.010 0.012 0.013 0.0087 0.006
0.61 0.28 0.18 0.11 0.07 0.09 0.12 0.08
THIOREDOXIN
REDUCTASE
ASSAY
613
The second method is the coupled reduction of insulin [or glutathione, or for E. coli thioredoxin reductase, 5,5’-dithiobis-(2nitrobenzoic acid) (DTNB)] in the presence of thioredoxin and NADPH. The major disadvantage of this method is that the sample must be free of other enzymes generating (or consuming) sulfhydryl groups. A supply of thioredoxin is also required. The third method which has been used with mammalian thioredoxin reductase is the direct reduction of DTNB. Although this method is quick and simple, the reaction rate is not linear with the concentration of the enzyme so that it is not a quantitative method. None of these methods is really satisfactory with crude samples, due to either nonspecificity or interference by contaminants. Here we introduce an immunochemical method which avoids some of the previous problems. This method is both sensitive and specific. The specificity depends on the antibody, which requires that a highly purified enzyme be used as the antigen. The previous methods require 10 to 100 ng of enzyme whereas this method requires only 3 to 10 ng. It is thus up to 10 times more sensitive. Of course the new method does not directly measure the enzyme activity; it measures antigen. With 0.3 mg of purified enzyme one can obtain more than 300 mg of antibody which can be used in thousands of assays. In order to economize on pure enzyme a less pure sample can be used for the standard curve in complement fixation, providing it is first calibrated with the pure enzyme. In Table 2 we see that thioredoxin reductase is most abundant in tumor and liver. The thioredoxin reductase prepared from liver has an electrophoretic mobility slightly different from that of the tumor enzyme (Chen et al., paper in preparation). The ratio of enzyme activity to complement-fixing antigen, however, differs by less than 20% between the two tissues. When a sample of enzyme was modified by reduction with 1% 2-mercaptoethanol or by carboxymethylation with iodoacetate, the enzyme activity was lost but the ability to form precipitin remained. This suggests that the regions controlling the antigenicity are more stable than the region of the enzyme active site. The enzyme activity, as measured by coupled reduction of glutathione, decreased to 35% in the presence of excess antibody. The low activity/antigen ratio in Step III of Table 1 may be due to denaturation of some of the enzyme without destruction of its antigenicity, with the subsequent puritication step removing the denatured enzyme. ACKNOWLEDGMENTS We wish to thank Dr. J. Y. Wu at Baylor Coliege of Medicine for the gift of some reagents as well as for some valuable discussions. This work was supported by grants to E. C. Moore from the National Cancer Institute (CA 04464) and the Robert A. Welch Foundation (G-455).
614
CHEN AND MOORE
REFERENCES 1. 2. 3. 4. 5.
Moore, E. C., Reichard, P., and Thelander, L. (1964) 3. Biol. Ckem. 239, 344.5-3452. Orr, M. D., and Vitols, E. (1966) Biockem. Biopkys. Res. Commun. 25, 109-115. Porque, P. G., Baldesten, A., and Reichard, P. (1970)J. Biol. Ckem. 245, 2363-2370. Larsson, A. (1973) Eur. J. Biockem. 35, 346-349. Engstriim, N. E., Holmgren, A., Larsson, A., and Siiderhall, S. (1974)J. Biol. Ckem. 249,205-210.
6. Moore, E. C. (1967) Biockem. Biopkys. Res. Commun. 29, 264-268. 7. Chen, C. C., McCall, B. L. B., and Moore, E. C. (1977) Prep. Biockem. 7, 165-177. 8. Levy, H. B., and Sober, H. A. (1960) Proc. Sot. Exp. Biol. Med. 103, 250-252. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)J. Bioi. Ckem. 193,265-27X
10. Levine, L. (1967) in Handbook of Experimental 707-719, Blackwelf Scientific, Oxford.
immunology
(Weir, D. M., ed.), pp.
