PROTEIN
EXPRESSION
AND
PURIFICATION
2, 63-65 (19%)
Rapid Purification of ,&Glucuronidase from Human Liver by Immunoaffinity Chromatography Employing Specific Murine Monoclonal IgG Binding to Tresyl-Activated Agarose’ Kang-Jey
Ho of Pathology,
University of Alabama at Birmingham, Center, Birmingham, Alabama 35233
Department Veterans Affairs
Medical
Received January
15, 1991, and in revised form March 29, 1991
W e have developed a simple, rapid method for purification of B-glucuronidase from human liver in order to facilitate the study of its biochemical structure and pathophysiologic roles in both cholelithiasis and carcinogenesis. The procedure includes the following steps: (1) liver homogenization, (2) 25-45% saturated ammonium sulfate fractionation, (3) heat denaturation, and (4) immunoaffinity chromatography employing murine anti-human &glucuronidase monoclonal IgG binding to tresyl-activated agarose. &Glucuronidase constitutes 1.3 mg per 100 g of wet liver tissue. The enzyme can be purified with a 1 0 % overall yield and overall purification of 5000-fold in a a-day cycle on a fairly large scale by the method described. Polyacrylamide gel electrophoresis indicated minor contaminants in the final product which could be further purified by protein blot0 1991 Academic Press. Inc. ting.
P-Glucuronidase is a ubiquitous lysosomal enzyme existing in most cells and tissue fluids in the human body (l-4). This enzyme seems to play an important role in the formation of pigment gallstones by deconjugation of bilirubin diglucuronide to form water-insoluble unconjugated bilirubin (4). It may also enhance the carcinogenic effect of certain chemicals by rendering them water-insoluble through hydrolysis of their glucuranic acid conjugates (5-7). A large-scale, rapid purification of human @-glucuronidase should facilitate the study of its biochemical properties and the aforementioned pathophysiologic roles.
1 This study was supported Research Fund.
in part by the Veterans Affairs Medical
1046-5928/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
and
Human P-glucuronidase has been purified from liver, urine, and placenta by chemical and chromatographic means with or without employing polyclonal antibodies (l-3). Such methods were time-consuming, the purity of final products was uncertain, and the yield was rather low. W e have initially obtained semipurified /3-glucuronidase from the human liver by ammonium sulfate fractionation and heat denaturation followed by various chromatographic procedures. The enzyme was then used to immunize BALB/c mice for production of specific monoclonal antibodies. The antibodies produced were in turn used to purify the enzyme by immunoaffinity chromatography. Described here are the final streamlined procedure and the results of the purification. The details of the initial purification procedure will be reported elsewhere. The final method for purification of hepatic P-glucuronidase is simple, fast, and capable of yielding a highly purified enzyme in a large quantity. MATERIALS
AND
METHODS
Materials. Human livers were obtained at autopsy through the Tissue Procurement Unit at the University of Alabama Hospital with the approval of the Institutional Review Board on Human Studies. Leupeptin, pepstatin, and phenylmethanesulfonyl fluoride (PMSF) were purchased from Boehringer-Mannheim Biochemicals (Indianapolis, IN). Coomassie brilliant blue G-250 protein assay kit and Bio-Rad P-6DG desalting columns were purchased from Bio-Rad Laboratories (Richmond, CA). Phenolphthalein &glucuronide was obtained from United States Biochemical Corp. (Cleveland, OH). Tresyl chloride (2,2,2-trifluoroethanesulfonyl chloride)-activated agarose was purchased from Schleicher & Schuell (Keene, NH). All buffer re63
Inc. reserved.
64
KANG-JEY HO
agents and other chemicals were analytical grade. BALB/c mouse hybridoma ceils lines producing monoclonal antibodies specific for human fl-glucuronidase were produced and maintained in our laboratories. Murine anti-human P-glucuronidase monoclonal IgG was purified with a rProtein A purification kit (Beckman Instruments, Inc., Fullerton, CA) from ascitic fluid obtained from BALB/c mice inoculated intraperitoneally with the hybridoma cell line (H07B6-2). Liver homogenization. Human liver (500 g) was homogenized in 2; vol of the extractant which was comtetraacetate (EDTA), posed of 2 m M ethylenediamine 10 m l-CYSTEINE, 1 PM leupeptin, 1 PM pepstatin, and 0.2 mM PMSF in 0.1 M Tris buffer, pH 7.5. Pepstatin and PMSF were prepared fresh in methanol before being added to the extractant. Homogenization was carried out in a PTA 20 Brinkmann homogenizer (Brinkmann Instruments, Inc., Westbury, NY) at high speed for 1 min. The homogenate was centrifuged at 3000g for 30 min. The residue was discarded. Ammonium sulfate fractionation. The supernatant of the liver homogenate was mixed well with 0.333 vol of saturated ammonium sulfate at room temperature in 0.1 M Tris buffer, pH 7.5, containing 2 m M EDTA and 10 mM L-cysteine to make a final 25% saturation of ammonium sulfate solution. The supernatant after centrifugation at 30008 for 30 min was mixed further with 0.364 vol (volume of 25% ammonium sulfate) of the same saturated ammonium sulfate solution to reach a final saturation of 45%. After centrifugation at 3000g for 30 min, the precipitate was redissolved in approximately 100 ml of 67 mM phosphate buffer, pH 7.2. Heat denaturation. The redissolved precipitate in 25-45% saturated ammonium sulfate fraction was heated at 56°C in a water bath-shaker for 5 to 10 min or until gross precipitate appeared. The supernatant obtained after centrifugation at 3000g for 30 min contained the undenatured enzyme. Monoclonal IgG immunoafinity column chromatography. Murine monoclonal IgG specific for human P-glucuronidase at a concentration of 2 mg/ml or higher was coupled to tresyl chloride-activated agarose (1 ml of gel per 2-6 mg of protein) in 0.1 M NaHCO, buffer, pH 8.5, containing 0.5 M NaCl. The coupling took place at room temperature for 2 h, followed by washing the gel with 10 vol of coupling buffer to remove any unreacted IgG and resuspension of the gel in 10 vol of 1.0 M ethanolamine, pH 8.5, for 1 h at room temperature to inactivate unreacted tresyl groups. The gel was further washed sequentially with coupling buffer, 0.2 M sodium acetate/O.5 M NaCl, pH 4.5,0.2 M NaHCOJ0.5 M NaCl, pH 8.5, 6 M guanidine hydrochloride, and then 67 m M phosphate buffer140 m M NaCl, pH 7.2. Twenty-five milliliters of the supernatant after the heat denaturation step was applied to each column (inside diameter
1.5 cm and length 20 cm) containing the above IgGagarose preequilibrated with 67 m M phosphate buffer/ 40 m M NaCl, pH 7.2. The solution after first pass usually still contained some unbound fl-glucuronidase. Second and third passes were often necessary for complete extraction of the enzyme. Human P-glucuronidase bound specifically to the IgG was then eluted with 5 ml of 6 M guanidine hydrochloride in the same phosphate buffer. The eluate (20 ml) was immediately desalted by passing through a Bio-Rad P-6DG desalting column (inside diameter 2.5 cm and height 20 cm) preequilibrated with the same phosphate buffer. All column chromatography was carried out at room temperature. Other procedures. The protein content and p-glucuronidase activity in the specimen after each step of purification were determined, respectively, by the Coomassie brilliant blue G-250 dye binding method (8) and by an enzyme assay employingphenolphthalein glucuronide as the substrate (9). Electrophoresis was carried out in a Protean II slab cell (Bio-Rad). The proteins separated after electrophoresis were transferred out of the gel onto a nitrocellulose membrane in a Trans-Blot SD semidry transfer cell (Bio-Rad).
RESULTS Recovery of protein and @-glucuronidase from human liver after various steps of purification is summarized in Table 1. At the initial step of ammonium sulfate fractionation, about 93% of the protein in the supernatant of liver homogenate was eliminated and 33% of the enzyme was retained in the 25-45% saturation fraction, resulting in a 4.6-fold purification. After heat denaturation, 97% of the protein in the original liver homogenate was removed and 20% of the enzyme activity was retained, resulting in an B-fold purification of the enzyme. About 10% of the enzyme activity and 0.002% of the protein in the original liver homogenate were recovered after monoclonal IgG immunoaffinity chromatography, representing a 5000-fold purification. Polyacrylamide gel electrophoresis of the final purified product under nondenaturing conditions showed a thick band of @-glucuronidase and two additional faint, fast moving bands. The enzyme isolated from the thick band after blotting had a specific activity not significantly different from that before electrophoresis.
DISCUSSION Human P-glucuronidase man liver and urine with cedures and ammonium were quite time-consuming the liver enzyme showed compared with the initial
has been purified from huvarious chromatographic prosulfate fractionation, which (1,2). The final product of only a 180-fold purification as crude extract (2). The purified
RAPID
PURIFICATION
OF /3-GLUCURONIDASE TABLE
Recovery
of Protein
and /3-Glucuronidase Protein bdd*
Procedure Crude extract of liver homogenate 25-45% Saturated ammonium sulfate fraction Heat denaturation Monoclonal IgG column ’ Mean f SD; number of determinations, b Per gram of starting material.
from
HUMAN
1
Human
Liver
after
Various
Enzyme activity (mmol/minlg)b
Steps
of Purification”
Specific activity (mmol/min/mg)
56.2 + 23.6 (100%)
0.85 f 0.31
4.6 f 1.2 (7%)
18.7 IL 10.3 (33%)
4.0 f 1.7 6.8 f 4.3 4260 f 2306
1.8 f 1.3 (3%) 0.013 t 0.009 (0.002%)
65
LIVER
65 f 30(100%)
11.4 -c 6.3 (20%)
5.4 + 2.2 (10%)
Purification (fold)
1 4.6 8.0 5000
12.
enzyme also had a low specific activity (1). Brot et al. (3), achieved a 360-fold purification of human placental P-glucuronidase with goat anti-rat preputial gland fl-glucuronidase polyclonal IgG affinity chromatography. We have achieved a 5000-fold purification of hepatic /3-glucuronidase with murine anti-human ,&glucuronidase monoclonal IgG affinity chromatography in this study. In our process the bulk of undesired tissue components was eliminated by initial centrifugation of the liver homogenate followed by 2545% saturated ammonium sulfate fractionation and then by heat denaturation at 56°C. The supernatant obtained after centrifugation of the heat-denatured sample was usually quite clear and ready for loading onto the IgG affinity column. Insufficiently heat-denatured specimens are cloudy and might clog the column and stop the flow. Tresyl-activated agarose is used for immobilization of amine- and thiol-containing ligands (10). Reaction of immunoglobulin with this gel results in the formation of a highly stable alkylamine bond and creation of an efficient affinity chromatography support. We have found that the commercially available tresyl-activated agarose (Schleicher & Schuell, Inc.) binds more immunoglobulin at a faster rate and higher efficiency than hydrazide gel. Since the average protein content of the purified P-glucuronidase was 1.3 pg per gram of wet liver tissue and only 10% of the original enzyme activity was recovered at the final step of purification (Table l), the hepatic content of fi-glucuronidase is estimated to be 0.013 mg/g wet tissue or 13 mg/lOOO g. Therefore, by our method we are capable of purifying 1 to 2 mg of /I-glucuronidase from 1 kg of liver in a 2-day cycle. urine
FROM
The efficiency of an immunoaffinity column depends largely upon the quantity of antibody coupling to it. The rate-limiting step in purification of hepatic &glucuronidase is thus the monoclonal antibody immunoaffinity chromatography. The coupling of our monoclonal antibodies to the tresyl-activated agarose was quite stable. Most of our columns have been used more than 50 times without losing their antigen (P-glucuronidase)-binding capacity. We should be able to extract most of the 20% of the original enzyme remaining in the supernatant after the heat denaturation step with the immunoaffinity chromatography provided that a sufficiently large quantity of the specific monoclonal antibody is available. Thus, the yield can be doubled. REFERENCES
1. Hygstedt,
O., and Jagenbury, 0. R. (1965) Can. J. Clin. Lab. Znvest. 17, 565-572. 2. Musa, B. U., Doe, R. P., and Seal, U. S. (1965) J. Biol. Chem. 240,
2811-2816. 3. Brot, F. E., Bell, C. E., Jr., and Sly, W. S. (1978) Biochemistry
17,
385-291.
4. Ho, K. J., Hsu, S. C., Chen, J. S., and Ho, L. H. (1986) Eur. J. Clin. Invest.
16,361-367.
5. Walaszek, Z., Hanausek-Walaszek,
M., Minton, J. P., and Webb, T. W. (1986) Curcinogenesis 7, 1463-1466. 6. Oredipe, 0. A., Barth, R. F., Hanausek-Walaszek, M., Sautins, J., Walasek, Z., and Webb, T. E. (1987) Cancer Z&t. 38.95-99. 7. Abou-Issa, H., Duruibe, V. A., Minton, J. P., Larroya, S., Dwivedi, C., and Webb, T. E. (1988) Proc. Natl. Acad. Sci. USA 85,4181-
4184. 8. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. 9. Ho, K. J., and Ho, L. H. C. (1980) Enzyme 25,361-370. 10. Nilsson, K., and Mosbach, K. (1984) in “Methods in Enzymology” (Jakoby, San Diego.
W. B., Ed.), Vol. 104, pp. 56-69, Academic
Press,
EXPRESSION
AND
PURIFICATION
2, 63-65 (19%)
Rapid Purification of ,&Glucuronidase from Human Liver by Immunoaffinity Chromatography Employing Specific Murine Monoclonal IgG Binding to Tresyl-Activated Agarose’ Kang-Jey
Ho of Pathology,
University of Alabama at Birmingham, Center, Birmingham, Alabama 35233
Department Veterans Affairs
Medical
Received January
15, 1991, and in revised form March 29, 1991
W e have developed a simple, rapid method for purification of B-glucuronidase from human liver in order to facilitate the study of its biochemical structure and pathophysiologic roles in both cholelithiasis and carcinogenesis. The procedure includes the following steps: (1) liver homogenization, (2) 25-45% saturated ammonium sulfate fractionation, (3) heat denaturation, and (4) immunoaffinity chromatography employing murine anti-human &glucuronidase monoclonal IgG binding to tresyl-activated agarose. &Glucuronidase constitutes 1.3 mg per 100 g of wet liver tissue. The enzyme can be purified with a 1 0 % overall yield and overall purification of 5000-fold in a a-day cycle on a fairly large scale by the method described. Polyacrylamide gel electrophoresis indicated minor contaminants in the final product which could be further purified by protein blot0 1991 Academic Press. Inc. ting.
P-Glucuronidase is a ubiquitous lysosomal enzyme existing in most cells and tissue fluids in the human body (l-4). This enzyme seems to play an important role in the formation of pigment gallstones by deconjugation of bilirubin diglucuronide to form water-insoluble unconjugated bilirubin (4). It may also enhance the carcinogenic effect of certain chemicals by rendering them water-insoluble through hydrolysis of their glucuranic acid conjugates (5-7). A large-scale, rapid purification of human @-glucuronidase should facilitate the study of its biochemical properties and the aforementioned pathophysiologic roles.
1 This study was supported Research Fund.
in part by the Veterans Affairs Medical
1046-5928/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
and
Human P-glucuronidase has been purified from liver, urine, and placenta by chemical and chromatographic means with or without employing polyclonal antibodies (l-3). Such methods were time-consuming, the purity of final products was uncertain, and the yield was rather low. W e have initially obtained semipurified /3-glucuronidase from the human liver by ammonium sulfate fractionation and heat denaturation followed by various chromatographic procedures. The enzyme was then used to immunize BALB/c mice for production of specific monoclonal antibodies. The antibodies produced were in turn used to purify the enzyme by immunoaffinity chromatography. Described here are the final streamlined procedure and the results of the purification. The details of the initial purification procedure will be reported elsewhere. The final method for purification of hepatic P-glucuronidase is simple, fast, and capable of yielding a highly purified enzyme in a large quantity. MATERIALS
AND
METHODS
Materials. Human livers were obtained at autopsy through the Tissue Procurement Unit at the University of Alabama Hospital with the approval of the Institutional Review Board on Human Studies. Leupeptin, pepstatin, and phenylmethanesulfonyl fluoride (PMSF) were purchased from Boehringer-Mannheim Biochemicals (Indianapolis, IN). Coomassie brilliant blue G-250 protein assay kit and Bio-Rad P-6DG desalting columns were purchased from Bio-Rad Laboratories (Richmond, CA). Phenolphthalein &glucuronide was obtained from United States Biochemical Corp. (Cleveland, OH). Tresyl chloride (2,2,2-trifluoroethanesulfonyl chloride)-activated agarose was purchased from Schleicher & Schuell (Keene, NH). All buffer re63
Inc. reserved.
64
KANG-JEY HO
agents and other chemicals were analytical grade. BALB/c mouse hybridoma ceils lines producing monoclonal antibodies specific for human fl-glucuronidase were produced and maintained in our laboratories. Murine anti-human P-glucuronidase monoclonal IgG was purified with a rProtein A purification kit (Beckman Instruments, Inc., Fullerton, CA) from ascitic fluid obtained from BALB/c mice inoculated intraperitoneally with the hybridoma cell line (H07B6-2). Liver homogenization. Human liver (500 g) was homogenized in 2; vol of the extractant which was comtetraacetate (EDTA), posed of 2 m M ethylenediamine 10 m l-CYSTEINE, 1 PM leupeptin, 1 PM pepstatin, and 0.2 mM PMSF in 0.1 M Tris buffer, pH 7.5. Pepstatin and PMSF were prepared fresh in methanol before being added to the extractant. Homogenization was carried out in a PTA 20 Brinkmann homogenizer (Brinkmann Instruments, Inc., Westbury, NY) at high speed for 1 min. The homogenate was centrifuged at 3000g for 30 min. The residue was discarded. Ammonium sulfate fractionation. The supernatant of the liver homogenate was mixed well with 0.333 vol of saturated ammonium sulfate at room temperature in 0.1 M Tris buffer, pH 7.5, containing 2 m M EDTA and 10 mM L-cysteine to make a final 25% saturation of ammonium sulfate solution. The supernatant after centrifugation at 30008 for 30 min was mixed further with 0.364 vol (volume of 25% ammonium sulfate) of the same saturated ammonium sulfate solution to reach a final saturation of 45%. After centrifugation at 3000g for 30 min, the precipitate was redissolved in approximately 100 ml of 67 mM phosphate buffer, pH 7.2. Heat denaturation. The redissolved precipitate in 25-45% saturated ammonium sulfate fraction was heated at 56°C in a water bath-shaker for 5 to 10 min or until gross precipitate appeared. The supernatant obtained after centrifugation at 3000g for 30 min contained the undenatured enzyme. Monoclonal IgG immunoafinity column chromatography. Murine monoclonal IgG specific for human P-glucuronidase at a concentration of 2 mg/ml or higher was coupled to tresyl chloride-activated agarose (1 ml of gel per 2-6 mg of protein) in 0.1 M NaHCO, buffer, pH 8.5, containing 0.5 M NaCl. The coupling took place at room temperature for 2 h, followed by washing the gel with 10 vol of coupling buffer to remove any unreacted IgG and resuspension of the gel in 10 vol of 1.0 M ethanolamine, pH 8.5, for 1 h at room temperature to inactivate unreacted tresyl groups. The gel was further washed sequentially with coupling buffer, 0.2 M sodium acetate/O.5 M NaCl, pH 4.5,0.2 M NaHCOJ0.5 M NaCl, pH 8.5, 6 M guanidine hydrochloride, and then 67 m M phosphate buffer140 m M NaCl, pH 7.2. Twenty-five milliliters of the supernatant after the heat denaturation step was applied to each column (inside diameter
1.5 cm and length 20 cm) containing the above IgGagarose preequilibrated with 67 m M phosphate buffer/ 40 m M NaCl, pH 7.2. The solution after first pass usually still contained some unbound fl-glucuronidase. Second and third passes were often necessary for complete extraction of the enzyme. Human P-glucuronidase bound specifically to the IgG was then eluted with 5 ml of 6 M guanidine hydrochloride in the same phosphate buffer. The eluate (20 ml) was immediately desalted by passing through a Bio-Rad P-6DG desalting column (inside diameter 2.5 cm and height 20 cm) preequilibrated with the same phosphate buffer. All column chromatography was carried out at room temperature. Other procedures. The protein content and p-glucuronidase activity in the specimen after each step of purification were determined, respectively, by the Coomassie brilliant blue G-250 dye binding method (8) and by an enzyme assay employingphenolphthalein glucuronide as the substrate (9). Electrophoresis was carried out in a Protean II slab cell (Bio-Rad). The proteins separated after electrophoresis were transferred out of the gel onto a nitrocellulose membrane in a Trans-Blot SD semidry transfer cell (Bio-Rad).
RESULTS Recovery of protein and @-glucuronidase from human liver after various steps of purification is summarized in Table 1. At the initial step of ammonium sulfate fractionation, about 93% of the protein in the supernatant of liver homogenate was eliminated and 33% of the enzyme was retained in the 25-45% saturation fraction, resulting in a 4.6-fold purification. After heat denaturation, 97% of the protein in the original liver homogenate was removed and 20% of the enzyme activity was retained, resulting in an B-fold purification of the enzyme. About 10% of the enzyme activity and 0.002% of the protein in the original liver homogenate were recovered after monoclonal IgG immunoaffinity chromatography, representing a 5000-fold purification. Polyacrylamide gel electrophoresis of the final purified product under nondenaturing conditions showed a thick band of @-glucuronidase and two additional faint, fast moving bands. The enzyme isolated from the thick band after blotting had a specific activity not significantly different from that before electrophoresis.
DISCUSSION Human P-glucuronidase man liver and urine with cedures and ammonium were quite time-consuming the liver enzyme showed compared with the initial
has been purified from huvarious chromatographic prosulfate fractionation, which (1,2). The final product of only a 180-fold purification as crude extract (2). The purified
RAPID
PURIFICATION
OF /3-GLUCURONIDASE TABLE
Recovery
of Protein
and /3-Glucuronidase Protein bdd*
Procedure Crude extract of liver homogenate 25-45% Saturated ammonium sulfate fraction Heat denaturation Monoclonal IgG column ’ Mean f SD; number of determinations, b Per gram of starting material.
from
HUMAN
1
Human
Liver
after
Various
Enzyme activity (mmol/minlg)b
Steps
of Purification”
Specific activity (mmol/min/mg)
56.2 + 23.6 (100%)
0.85 f 0.31
4.6 f 1.2 (7%)
18.7 IL 10.3 (33%)
4.0 f 1.7 6.8 f 4.3 4260 f 2306
1.8 f 1.3 (3%) 0.013 t 0.009 (0.002%)
65
LIVER
65 f 30(100%)
11.4 -c 6.3 (20%)
5.4 + 2.2 (10%)
Purification (fold)
1 4.6 8.0 5000
12.
enzyme also had a low specific activity (1). Brot et al. (3), achieved a 360-fold purification of human placental P-glucuronidase with goat anti-rat preputial gland fl-glucuronidase polyclonal IgG affinity chromatography. We have achieved a 5000-fold purification of hepatic /3-glucuronidase with murine anti-human ,&glucuronidase monoclonal IgG affinity chromatography in this study. In our process the bulk of undesired tissue components was eliminated by initial centrifugation of the liver homogenate followed by 2545% saturated ammonium sulfate fractionation and then by heat denaturation at 56°C. The supernatant obtained after centrifugation of the heat-denatured sample was usually quite clear and ready for loading onto the IgG affinity column. Insufficiently heat-denatured specimens are cloudy and might clog the column and stop the flow. Tresyl-activated agarose is used for immobilization of amine- and thiol-containing ligands (10). Reaction of immunoglobulin with this gel results in the formation of a highly stable alkylamine bond and creation of an efficient affinity chromatography support. We have found that the commercially available tresyl-activated agarose (Schleicher & Schuell, Inc.) binds more immunoglobulin at a faster rate and higher efficiency than hydrazide gel. Since the average protein content of the purified P-glucuronidase was 1.3 pg per gram of wet liver tissue and only 10% of the original enzyme activity was recovered at the final step of purification (Table l), the hepatic content of fi-glucuronidase is estimated to be 0.013 mg/g wet tissue or 13 mg/lOOO g. Therefore, by our method we are capable of purifying 1 to 2 mg of /I-glucuronidase from 1 kg of liver in a 2-day cycle. urine
FROM
The efficiency of an immunoaffinity column depends largely upon the quantity of antibody coupling to it. The rate-limiting step in purification of hepatic &glucuronidase is thus the monoclonal antibody immunoaffinity chromatography. The coupling of our monoclonal antibodies to the tresyl-activated agarose was quite stable. Most of our columns have been used more than 50 times without losing their antigen (P-glucuronidase)-binding capacity. We should be able to extract most of the 20% of the original enzyme remaining in the supernatant after the heat denaturation step with the immunoaffinity chromatography provided that a sufficiently large quantity of the specific monoclonal antibody is available. Thus, the yield can be doubled. REFERENCES
1. Hygstedt,
O., and Jagenbury, 0. R. (1965) Can. J. Clin. Lab. Znvest. 17, 565-572. 2. Musa, B. U., Doe, R. P., and Seal, U. S. (1965) J. Biol. Chem. 240,
2811-2816. 3. Brot, F. E., Bell, C. E., Jr., and Sly, W. S. (1978) Biochemistry
17,
385-291.
4. Ho, K. J., Hsu, S. C., Chen, J. S., and Ho, L. H. (1986) Eur. J. Clin. Invest.
16,361-367.
5. Walaszek, Z., Hanausek-Walaszek,
M., Minton, J. P., and Webb, T. W. (1986) Curcinogenesis 7, 1463-1466. 6. Oredipe, 0. A., Barth, R. F., Hanausek-Walaszek, M., Sautins, J., Walasek, Z., and Webb, T. E. (1987) Cancer Z&t. 38.95-99. 7. Abou-Issa, H., Duruibe, V. A., Minton, J. P., Larroya, S., Dwivedi, C., and Webb, T. E. (1988) Proc. Natl. Acad. Sci. USA 85,4181-
4184. 8. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. 9. Ho, K. J., and Ho, L. H. C. (1980) Enzyme 25,361-370. 10. Nilsson, K., and Mosbach, K. (1984) in “Methods in Enzymology” (Jakoby, San Diego.
W. B., Ed.), Vol. 104, pp. 56-69, Academic
Press,
