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Chemical synthesis of [32P]pyrophosphate with high specific activity
ANALYTICAL
BIOCHEMISTRY
Chemical
108, 279-281 (1980)
Synthesis
of [32P]Pyrophosphate KIT
McArdle
Laboratory
for Cancer
S. LAM
Research,
AND CHARLES
The University
with High Specific
Activity1
B. KASPER
of Wisconsin-Madison,
Madison,
Wisconsin
53706
Received June 2, 1980 A simple procedure for synthesizing [32P]pyrophosphate with high specific activity (about 100 Ci/mmol) is described. [32P]Pyrophosphate was formed by simple dehydration of inorganic [3*P]phosphate and subsequently purified by DEAE-Sephadex A-25 chromatography. The yield was 20-40%.
Pyrophosphate is generated in a wide variety of biochemical reactions. A large quantity of pyrophosphate is produced during DNA or RNA synthesis. Another major group of enzymes that produces pyrophosphate is the ligases (synthetases), which catalyze the joining of two molecules with concomitant hydrolysis of ATP to AMP and pyrophosphate. Examples of this group of enzymes are the aminoacyl-tRNA synthetases, arylCoA synthetase, amide synthetase and peptide synthetases (1). In addition, there are hydrolytic enzymes that simply hydrolyze nucleoside triphosphates to nucleoside monophosphate and pyrophosphate (e.g., ATP pyrophosphatase and nucleoside-triphosphate pyrophosphatase) (1). [32P]Pyrophosphate (PPJ has been a valuable tool for kinetics studies on some of these enzymes (2,3). In recent years, several enzymes utilizing pyrophosphate as the energy source have been described (45): carboxytransphosphorylase; pyruvate, phosphate dikinase; pyrophosphate-phosphofructokinase and pyrophosphate-acetate kinase. We recently described a novel pyrophosphate:protein phosphotransferase, which utilizes pyrophosphate as the phosphoryl donor for the ’ From the McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wise. 53706. Supported by Grants CA-17300, CAO-07175, and CA23076 from the National Cancer Institute. 279
selective phosphorylation of two microsomal polypeptides (6,7). To study the protein phosphorylation reaction, [32P]pyrophosphate with high specific activity is required (7). Commercially available [32P]pyrophosphate is inadequate because of its relatively low specific activity (0.1-4 Cilmmol). In this paper, we shall describe a simple procedure for synthesizing [32P]pyrophosphate with a specific activity in the range of 50-150 Ci/mmol. MATERIALS
AND METHODS
Carrier-free [32P]orthophosphoric acid (in 0.02 N HCl) was purchased from New England Nuclear. NaH,PO, (0.4 pmol) contained in 0.5 ml was added to 15 mCi [32Plpi and carefully transferred into a siliconized Kimax test tube (16 x 125 mm). The mixture was maintained at 200°C in a heating bath (NaNO,:NaNO,:KNO, in a ratio of 40:7:53, Ref. (8)) until completely dry. The test tube was cooled and 0.5 ml doubly distilled water was added. The heating and cooling process was repeated four times. After the test tube cooled, the reaction products were dissolved in 1 ml of 0.4 mM NaH,PO, and applied to a DEAE-Sephadex A-25 column (1 x 20 cm) equilibrated with 0.1 M ammonium acetate (pH 7.0). A linear gradient (200 ml) of 0.1 to 1.0 M ammonium acetate was used to develop the column. 0003-2697/80/160279-03$02.00/O Copyright All rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
280
LAM AND KASPER
14
16
18
20
22
24
26
28
FRACTION
30
32
34
36
38
40
Rxn
NUMBER
FIG. 1. Autoradiographic analysis of chemically synthesized pyrophosphate separated by thin-layer chromatography. Rxn: reaction mixture containing P,, PP,, and a small amount of an unidentified compound, probably tripolyphosphate (PJn. The reaction mixture was loaded onto a DEAE-Sephadex column and Pi and PPi were eluted by a linear gradient of 0.1- 1 M ammonium acetate (pH 7.0). See Fig. 2 for quantitative analysis.
The flow rate was about 40 ml/h, and 4-ml fractions were collected. Five microliters of each fraction was mixed with 50 ~1 of a mixture of 0.5 mM tetrasodium pyrophosphate and 0.5 mM NaH,PO,. One microliter of the final mixture was carefully spotted onto a polyethyleneimine-impregnated cellulose plate (Brinkman Instruments, Inc.), and ascending thin-layer chromatography was run in 1.8, d LiCl, 0.01 M EDTA, pH 6.5. The dried chromatogram was placed next to Kodak X-Omat R film and exposed for about 1 h. Fractions containing [32P]PPi were pooled and ammonium acetate was 14
14 pi 12-
-1.21 I
IOv I 6o_ i B6 .
4.’
.’
/’
/
.’
/’
.’
.’
.’
l’
.’
I’
/’
‘O ; -O..$
P Y
-06< 3
PPi -a2
FRACTION
NUMBER
FIG. 2. Purification of [32PlpPi. The reaction mixture was loaded onto a DEAE-Sephadex A-25 column and a linear gradient of 0.1 to 1 M ammonium acetate (pH 7.0) was used to develop the column. An aliquot of each column fraction was counted by liquid scintillation. Refer to Fig. 1 for the autoradiographic analysis.
removed by either(i) drying by rotary evaporation at 50°C under vacuum with several washes of double distilled water, or (ii) shell freezing the pooled fractions and lyophilizing under high vacuum with several washes of double distilled water. RESULTS AND DISCUSSION
The reaction products after the four multiple dehydration steps are shown in Fig. 1 (Rxn). The yield of [32P]pyrophosphate was about 35%. Another product (Pi),, probably tripolyphosphate, was also detected but only in small amounts. A low but significant level of 32P-labeled product(s) also remained at the origin of the thin-layer chromatogram. These are probably polyphosphates of higher molecular weights. DEAE-Sephadex A-25 provides a very good separation for Pi, PPi, and (PJn (Figs. 1 and 2). Inorganic phosphate and PPi were eluted at approximately 0.4 and 0.6 M ammonium acetate (pH 7.0), respectively (Fig. 2). [32P]Pyrophosphate is stable in aqueous solution at room temperature for several days; however, for longer periods of time it is routinely stored at -20°C. Only a small amount of [“‘PIPPi was formed during the first two dehydration processes (data not shown). This is probably due to the presence of a minute amount of HCl still remaining from the original [““PIorthophosphoric acid mix. After the third
[“PIPYROPHOSPHATE
heating process, the yield of [32P]PPi remained relatively constant up to eight heating cycles. A higher temperature generated more polyphosphate but did not increase the yield of [32PlpPi. Therefore, four heating cycles at 200°C were routinely used. Because carrier-free [32P]Pi was used, the specific activity of the final [32P]PPi depended only on the amount of unlabeled NaH,PO, added. Using the amount of NaH,PO, added before dehydration, the specific activity of the final purified [32P]PPi can be easily calculated. The specific activity is exactly twice that of the NaH232P0, before dehydration. Theoretically, carrierfree [32P]PPi can be synthesized by this procedure. We hope that this simple and inexpensive procedure will prove useful to other investigators studying the biochemistry of [32P]PPi.
281
SYNTHESIS
REFERENCES 1. International Union of Biochemistry (1979) Enzyme Nomenclature, pp. 220-227, Academic Press, New York. 2. Kern, D., and Cierge, R. (1979) FEBS Letr. 103, 274-28 I. 3. Zinoviev, V. V., Rubtsova, N. G., Lawrik, 0. I., Malygin, E. G., Akhveroyan, V. Z.. Favorova, D. O., and Kisselev, L. L. (1977) FEBS Let?. 82, 130- 134. 4. Reeves, R. E. (1976) Trends Biochem. Sci. 1, 53-55. 5. Wood, H. G., O’Brien, W. E., and Michaels, G. (1977) Advan. Enzymol. 45,85-155. 6. Lam, K. S., and Kasper, C. B. (1980) J. BioL Chem. 255259-266. 7. Lam, K. S., and Kasper, C. B. (1980) Proc. Nat. Acad. Sci. USA 71, 1927-1931. 8. Gordon. A. J., and Ford, R. A. ( 1972) The Chemist’s Companion: A Handbook of Practical Data, Techniques, and References, p. 450, Wiley, New York.
BIOCHEMISTRY
Chemical
108, 279-281 (1980)
Synthesis
of [32P]Pyrophosphate KIT
McArdle
Laboratory
for Cancer
S. LAM
Research,
AND CHARLES
The University
with High Specific
Activity1
B. KASPER
of Wisconsin-Madison,
Madison,
Wisconsin
53706
Received June 2, 1980 A simple procedure for synthesizing [32P]pyrophosphate with high specific activity (about 100 Ci/mmol) is described. [32P]Pyrophosphate was formed by simple dehydration of inorganic [3*P]phosphate and subsequently purified by DEAE-Sephadex A-25 chromatography. The yield was 20-40%.
Pyrophosphate is generated in a wide variety of biochemical reactions. A large quantity of pyrophosphate is produced during DNA or RNA synthesis. Another major group of enzymes that produces pyrophosphate is the ligases (synthetases), which catalyze the joining of two molecules with concomitant hydrolysis of ATP to AMP and pyrophosphate. Examples of this group of enzymes are the aminoacyl-tRNA synthetases, arylCoA synthetase, amide synthetase and peptide synthetases (1). In addition, there are hydrolytic enzymes that simply hydrolyze nucleoside triphosphates to nucleoside monophosphate and pyrophosphate (e.g., ATP pyrophosphatase and nucleoside-triphosphate pyrophosphatase) (1). [32P]Pyrophosphate (PPJ has been a valuable tool for kinetics studies on some of these enzymes (2,3). In recent years, several enzymes utilizing pyrophosphate as the energy source have been described (45): carboxytransphosphorylase; pyruvate, phosphate dikinase; pyrophosphate-phosphofructokinase and pyrophosphate-acetate kinase. We recently described a novel pyrophosphate:protein phosphotransferase, which utilizes pyrophosphate as the phosphoryl donor for the ’ From the McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wise. 53706. Supported by Grants CA-17300, CAO-07175, and CA23076 from the National Cancer Institute. 279
selective phosphorylation of two microsomal polypeptides (6,7). To study the protein phosphorylation reaction, [32P]pyrophosphate with high specific activity is required (7). Commercially available [32P]pyrophosphate is inadequate because of its relatively low specific activity (0.1-4 Cilmmol). In this paper, we shall describe a simple procedure for synthesizing [32P]pyrophosphate with a specific activity in the range of 50-150 Ci/mmol. MATERIALS
AND METHODS
Carrier-free [32P]orthophosphoric acid (in 0.02 N HCl) was purchased from New England Nuclear. NaH,PO, (0.4 pmol) contained in 0.5 ml was added to 15 mCi [32Plpi and carefully transferred into a siliconized Kimax test tube (16 x 125 mm). The mixture was maintained at 200°C in a heating bath (NaNO,:NaNO,:KNO, in a ratio of 40:7:53, Ref. (8)) until completely dry. The test tube was cooled and 0.5 ml doubly distilled water was added. The heating and cooling process was repeated four times. After the test tube cooled, the reaction products were dissolved in 1 ml of 0.4 mM NaH,PO, and applied to a DEAE-Sephadex A-25 column (1 x 20 cm) equilibrated with 0.1 M ammonium acetate (pH 7.0). A linear gradient (200 ml) of 0.1 to 1.0 M ammonium acetate was used to develop the column. 0003-2697/80/160279-03$02.00/O Copyright All rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
280
LAM AND KASPER
14
16
18
20
22
24
26
28
FRACTION
30
32
34
36
38
40
Rxn
NUMBER
FIG. 1. Autoradiographic analysis of chemically synthesized pyrophosphate separated by thin-layer chromatography. Rxn: reaction mixture containing P,, PP,, and a small amount of an unidentified compound, probably tripolyphosphate (PJn. The reaction mixture was loaded onto a DEAE-Sephadex column and Pi and PPi were eluted by a linear gradient of 0.1- 1 M ammonium acetate (pH 7.0). See Fig. 2 for quantitative analysis.
The flow rate was about 40 ml/h, and 4-ml fractions were collected. Five microliters of each fraction was mixed with 50 ~1 of a mixture of 0.5 mM tetrasodium pyrophosphate and 0.5 mM NaH,PO,. One microliter of the final mixture was carefully spotted onto a polyethyleneimine-impregnated cellulose plate (Brinkman Instruments, Inc.), and ascending thin-layer chromatography was run in 1.8, d LiCl, 0.01 M EDTA, pH 6.5. The dried chromatogram was placed next to Kodak X-Omat R film and exposed for about 1 h. Fractions containing [32P]PPi were pooled and ammonium acetate was 14
14 pi 12-
-1.21 I
IOv I 6o_ i B6 .
4.’
.’
/’
/
.’
/’
.’
.’
.’
l’
.’
I’
/’
‘O ; -O..$
P Y
-06< 3
PPi -a2
FRACTION
NUMBER
FIG. 2. Purification of [32PlpPi. The reaction mixture was loaded onto a DEAE-Sephadex A-25 column and a linear gradient of 0.1 to 1 M ammonium acetate (pH 7.0) was used to develop the column. An aliquot of each column fraction was counted by liquid scintillation. Refer to Fig. 1 for the autoradiographic analysis.
removed by either(i) drying by rotary evaporation at 50°C under vacuum with several washes of double distilled water, or (ii) shell freezing the pooled fractions and lyophilizing under high vacuum with several washes of double distilled water. RESULTS AND DISCUSSION
The reaction products after the four multiple dehydration steps are shown in Fig. 1 (Rxn). The yield of [32P]pyrophosphate was about 35%. Another product (Pi),, probably tripolyphosphate, was also detected but only in small amounts. A low but significant level of 32P-labeled product(s) also remained at the origin of the thin-layer chromatogram. These are probably polyphosphates of higher molecular weights. DEAE-Sephadex A-25 provides a very good separation for Pi, PPi, and (PJn (Figs. 1 and 2). Inorganic phosphate and PPi were eluted at approximately 0.4 and 0.6 M ammonium acetate (pH 7.0), respectively (Fig. 2). [32P]Pyrophosphate is stable in aqueous solution at room temperature for several days; however, for longer periods of time it is routinely stored at -20°C. Only a small amount of [“‘PIPPi was formed during the first two dehydration processes (data not shown). This is probably due to the presence of a minute amount of HCl still remaining from the original [““PIorthophosphoric acid mix. After the third
[“PIPYROPHOSPHATE
heating process, the yield of [32P]PPi remained relatively constant up to eight heating cycles. A higher temperature generated more polyphosphate but did not increase the yield of [32PlpPi. Therefore, four heating cycles at 200°C were routinely used. Because carrier-free [32P]Pi was used, the specific activity of the final [32P]PPi depended only on the amount of unlabeled NaH,PO, added. Using the amount of NaH,PO, added before dehydration, the specific activity of the final purified [32P]PPi can be easily calculated. The specific activity is exactly twice that of the NaH232P0, before dehydration. Theoretically, carrierfree [32P]PPi can be synthesized by this procedure. We hope that this simple and inexpensive procedure will prove useful to other investigators studying the biochemistry of [32P]PPi.
281
SYNTHESIS
REFERENCES 1. International Union of Biochemistry (1979) Enzyme Nomenclature, pp. 220-227, Academic Press, New York. 2. Kern, D., and Cierge, R. (1979) FEBS Letr. 103, 274-28 I. 3. Zinoviev, V. V., Rubtsova, N. G., Lawrik, 0. I., Malygin, E. G., Akhveroyan, V. Z.. Favorova, D. O., and Kisselev, L. L. (1977) FEBS Let?. 82, 130- 134. 4. Reeves, R. E. (1976) Trends Biochem. Sci. 1, 53-55. 5. Wood, H. G., O’Brien, W. E., and Michaels, G. (1977) Advan. Enzymol. 45,85-155. 6. Lam, K. S., and Kasper, C. B. (1980) J. BioL Chem. 255259-266. 7. Lam, K. S., and Kasper, C. B. (1980) Proc. Nat. Acad. Sci. USA 71, 1927-1931. 8. Gordon. A. J., and Ford, R. A. ( 1972) The Chemist’s Companion: A Handbook of Practical Data, Techniques, and References, p. 450, Wiley, New York.