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A simple and sensitive method for detection of l -asparaginase by polyacrylamide gel electrophoresis
.4x ~I.Y’I’ICAL
HLOCHEMISTRY
50, 317-320
(1972)
A Simple and Sensitive Method for Detection of L-Asparaginase by Polyacrylamide G e l Electrophoresis
n-Asparaginase (n-asparagine amidohydrolase, EC 3.5.1.1) is an enzyme which has been applied as a potent antiproliferative drug in neoplastic diseases, particularly in leukemias. The enzyme was demonstrated to be most effective in the therapy of acute lymphoblastic leukemia (1). Most clinical studies employ the enzyme isolated from Escherichia coli. Two forms (designated EC-l and EC-2) of n-asparaginase, only one (EC-2) of which is effective in the treatment of leukemia, were found in strains of E. coli (2). The two forms differ in solubility in ammonium sulfate solution, chromatographic properties, and effect of pH on the enzyme activity. Isoenzymic patterns of E. coli L-asparaginase were also investigated by acrylamide gel electrophoresis. However, the method involved gel slicing and determination of enzyme activity in individual gel sections (5). A method for detecting this enzyme in the gel was, therefore, desirable. The present communication describes such a simple and sensitive procedure. The procedure is based upon the forming of insoluble complex of sodium tetraphenylboronNaB (C&H,) ,-with NH,+ ion (4). Materials and Methods. Serratia marcescens PCM 501 was obtained from the Polish Collection of Microorganisms, Wroclaw, and E. coli 055 B5 was our laboratory strain. Microorganisms were grown as previously described (7). Cells were washed in distilled water, suspended in 0.1% Cetavlon (Cetyltrimethylammonium bromide, Chemapol, Prague, Czechoslovakia), and disrupted by freezing and thawing (repeated five times). After centrifugation L-asparaginase activity (8) and protein content (6) were measured in crude extracts. Purified E. coli n-asparaginase (Crasnitin) was obtained from Bayer. Sodium tetraphenylboron-NaB (C,H,) 4-was obtained from the Institute of Organic Chemistry, PAN, Warszawa, and was recrystallized twice from ether, before use. Recrystallization procedure: the reagent was dissolved in 317 Copyright @ 1972 by Academic Press. Inc. All rights of reproduction in any form reserved.
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ether at -10°C and filtered, and the filtrate left at room temperature for crystallization. Experimental Procedure. Six 83 X 7 mm glass tubes, with Plexiglas rod (100 :K 3 mm) held vertically in the center of each tube, were fixed in a Plexiglas frame. Gel was polymerized and disc electrophoresis performed generally according to the procedure of Clark (3). Appropriate dilutions of the E. wli and S. marcescens crude extracts and of Crasnitin were prepared in 10% sucrose so that L-asparaginase activities were 0.1 NJ/ml. Then 100 ~1 of each sample was submitted to electrophoresis on polyacrylamide gels with Plexiglas rods in the center. Following electrophoresis, the separation tubes with gel were mounted back in the Plexiglas frame. The Plexiglas rods were pulled out and the lumens filled with a mixture of equal volumes of melted 2% purified Agar-Difco (2% purified Agar-Difco in twice-distilled water) and of substrate sodium tetraphenylboron solution (2.6 mg L-asparagine, 3.5 mg sodium tetraphenylboron, and 1 ml 0.2 M Tris-phosphate buffer, pH 8.0). Before mixing, both parts of the mixture were kept in a 45°C water bath.
FIO. 1. Detection of L-asparaginase after gel electrophoresis: (A) crude extract of E. cola 055 B5 (protein 10 cg, enzyme activity 0.01 III); (Cl crude extract of S. marcescens PCM 501 (protein 35 pg, enzyme activity 0.01 III); (B) mixture of A and C; (D) Crasnitin (Bayer preparation of L-asparaginase, protein 0.1 gg, enzyme activity 0.01 III) ; (E) Crasnitin (protein 100 pg). A-D, gels ‘
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319
After the agar had solidified, the gels were overlayered with distilled water to prevent evaporation and were incubated at 37°C for 30 min or more until white bands were visually discernible in the agar gel. Alternatively, separation was carried out on polyacrylamide gel slabs. After electrophoresis the incubation mixture described above was simply poured on the surface of the slab. Then the acrylamide-agar gel %andwith” was incubated at 37°C in a humid chamber. To stop reaction the gel cylinders or slabs were immersed in 7% acetic acid. Photographs were taken on a black background. Results and Comments. The method described here is sensitive and convenient. It permits the detection (without the necessity of gel slicing) of as low enzyme activity as 0.001 IU (0.01 pg Crasnitin). Both purified enzyme and crude cell extracts may be examined for L-asparaginase activity (Fig. 1). Use of this method has enabled us to
FIG. 2. Polyacrylamide slab (Crasnitin): (A) protein 160 (B) Crasnitin protein 1 gg, activity. Both A and B were
electrophoresis of Bayer preparation of L-asparaginase pg, enzyme activity 16 IU, stained with amido black; enzyme activity 0.1 IU, stained for L-asparaginase run in duplicate.
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confirm Ho and Milikin’s (5) observation on the existence of multiple forms of L-asparaginase directly in the gel (Fig. 2). Furthermore, the method seems to be readily adaptable to other ammonia-liberating enzymes by simply incorporating the reagents used for their spectrophotometric assay into agar gel containing sodium tetraphenylboron. REFERENCES 1. BERNARD, J., AND B~IRON, M. (1970) Seminars Hemat. 7, 427. 2. CAMPBELL, H. A., MASHBRUN, L. T., BOYSE, E. A., AND OLD, L. J. (1967) Biochemistry 6, 721. 3. CLARKE, J. T. (1964) Ann. N. Y. Acud. Sci. 121, 428. 4. Gmss, G. H. (1953) Chemist Analyst 42, 50. 5. Ho, P. P. K., AND MILIKIN, E. B. (1970) B&him. Biophgs. Acta 206, 196. 6. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265. (Poland) 23, 207. 7. PAJDAK, E. (1971) Ezp. Med. Microbial. 8. ROBERTS,J., BURSON, G., AND HILL, J. M. (1968) J. Bacterial. 95, 2117.
EL~BIETA PAJDAK W~ADYS~AW PAJDAK Microbiological Laboratory of Pediatrics Institute School of Medicine, Cracow,
Polclnd
of Clinical Chemistry Department Institute of Internal Medicine School of Medicine, Cracow, Poland Received March lY, 19Yd Accepted June 1, 19773
HLOCHEMISTRY
50, 317-320
(1972)
A Simple and Sensitive Method for Detection of L-Asparaginase by Polyacrylamide G e l Electrophoresis
n-Asparaginase (n-asparagine amidohydrolase, EC 3.5.1.1) is an enzyme which has been applied as a potent antiproliferative drug in neoplastic diseases, particularly in leukemias. The enzyme was demonstrated to be most effective in the therapy of acute lymphoblastic leukemia (1). Most clinical studies employ the enzyme isolated from Escherichia coli. Two forms (designated EC-l and EC-2) of n-asparaginase, only one (EC-2) of which is effective in the treatment of leukemia, were found in strains of E. coli (2). The two forms differ in solubility in ammonium sulfate solution, chromatographic properties, and effect of pH on the enzyme activity. Isoenzymic patterns of E. coli L-asparaginase were also investigated by acrylamide gel electrophoresis. However, the method involved gel slicing and determination of enzyme activity in individual gel sections (5). A method for detecting this enzyme in the gel was, therefore, desirable. The present communication describes such a simple and sensitive procedure. The procedure is based upon the forming of insoluble complex of sodium tetraphenylboronNaB (C&H,) ,-with NH,+ ion (4). Materials and Methods. Serratia marcescens PCM 501 was obtained from the Polish Collection of Microorganisms, Wroclaw, and E. coli 055 B5 was our laboratory strain. Microorganisms were grown as previously described (7). Cells were washed in distilled water, suspended in 0.1% Cetavlon (Cetyltrimethylammonium bromide, Chemapol, Prague, Czechoslovakia), and disrupted by freezing and thawing (repeated five times). After centrifugation L-asparaginase activity (8) and protein content (6) were measured in crude extracts. Purified E. coli n-asparaginase (Crasnitin) was obtained from Bayer. Sodium tetraphenylboron-NaB (C,H,) 4-was obtained from the Institute of Organic Chemistry, PAN, Warszawa, and was recrystallized twice from ether, before use. Recrystallization procedure: the reagent was dissolved in 317 Copyright @ 1972 by Academic Press. Inc. All rights of reproduction in any form reserved.
318
SHORT
COMMUNICATIONS
ether at -10°C and filtered, and the filtrate left at room temperature for crystallization. Experimental Procedure. Six 83 X 7 mm glass tubes, with Plexiglas rod (100 :K 3 mm) held vertically in the center of each tube, were fixed in a Plexiglas frame. Gel was polymerized and disc electrophoresis performed generally according to the procedure of Clark (3). Appropriate dilutions of the E. wli and S. marcescens crude extracts and of Crasnitin were prepared in 10% sucrose so that L-asparaginase activities were 0.1 NJ/ml. Then 100 ~1 of each sample was submitted to electrophoresis on polyacrylamide gels with Plexiglas rods in the center. Following electrophoresis, the separation tubes with gel were mounted back in the Plexiglas frame. The Plexiglas rods were pulled out and the lumens filled with a mixture of equal volumes of melted 2% purified Agar-Difco (2% purified Agar-Difco in twice-distilled water) and of substrate sodium tetraphenylboron solution (2.6 mg L-asparagine, 3.5 mg sodium tetraphenylboron, and 1 ml 0.2 M Tris-phosphate buffer, pH 8.0). Before mixing, both parts of the mixture were kept in a 45°C water bath.
FIO. 1. Detection of L-asparaginase after gel electrophoresis: (A) crude extract of E. cola 055 B5 (protein 10 cg, enzyme activity 0.01 III); (Cl crude extract of S. marcescens PCM 501 (protein 35 pg, enzyme activity 0.01 III); (B) mixture of A and C; (D) Crasnitin (Bayer preparation of L-asparaginase, protein 0.1 gg, enzyme activity 0.01 III) ; (E) Crasnitin (protein 100 pg). A-D, gels ‘
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319
After the agar had solidified, the gels were overlayered with distilled water to prevent evaporation and were incubated at 37°C for 30 min or more until white bands were visually discernible in the agar gel. Alternatively, separation was carried out on polyacrylamide gel slabs. After electrophoresis the incubation mixture described above was simply poured on the surface of the slab. Then the acrylamide-agar gel %andwith” was incubated at 37°C in a humid chamber. To stop reaction the gel cylinders or slabs were immersed in 7% acetic acid. Photographs were taken on a black background. Results and Comments. The method described here is sensitive and convenient. It permits the detection (without the necessity of gel slicing) of as low enzyme activity as 0.001 IU (0.01 pg Crasnitin). Both purified enzyme and crude cell extracts may be examined for L-asparaginase activity (Fig. 1). Use of this method has enabled us to
FIG. 2. Polyacrylamide slab (Crasnitin): (A) protein 160 (B) Crasnitin protein 1 gg, activity. Both A and B were
electrophoresis of Bayer preparation of L-asparaginase pg, enzyme activity 16 IU, stained with amido black; enzyme activity 0.1 IU, stained for L-asparaginase run in duplicate.
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confirm Ho and Milikin’s (5) observation on the existence of multiple forms of L-asparaginase directly in the gel (Fig. 2). Furthermore, the method seems to be readily adaptable to other ammonia-liberating enzymes by simply incorporating the reagents used for their spectrophotometric assay into agar gel containing sodium tetraphenylboron. REFERENCES 1. BERNARD, J., AND B~IRON, M. (1970) Seminars Hemat. 7, 427. 2. CAMPBELL, H. A., MASHBRUN, L. T., BOYSE, E. A., AND OLD, L. J. (1967) Biochemistry 6, 721. 3. CLARKE, J. T. (1964) Ann. N. Y. Acud. Sci. 121, 428. 4. Gmss, G. H. (1953) Chemist Analyst 42, 50. 5. Ho, P. P. K., AND MILIKIN, E. B. (1970) B&him. Biophgs. Acta 206, 196. 6. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265. (Poland) 23, 207. 7. PAJDAK, E. (1971) Ezp. Med. Microbial. 8. ROBERTS,J., BURSON, G., AND HILL, J. M. (1968) J. Bacterial. 95, 2117.
EL~BIETA PAJDAK W~ADYS~AW PAJDAK Microbiological Laboratory of Pediatrics Institute School of Medicine, Cracow,
Polclnd
of Clinical Chemistry Department Institute of Internal Medicine School of Medicine, Cracow, Poland Received March lY, 19Yd Accepted June 1, 19773