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[24b] Biosynthetic preparation of 32P-labeled nucleoside 5′-phosphates and derivatives
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[24b] B i o s y n t h e t i c P r e p a r a t i o n of 3 2 p - L a b e l e d N u c l e o s i d e 5'-Phosphates and Derivatives 1
By ROBERT B.
HURLBERT and N. BURR FURLONG
Introduction A method s is described for the biosynthetic and enzymatic preparation of CTP, ATP, GTP, UTP, CDP, dATP, dCTP, dGTP, and TTP labeled with 32p in the alpha position. Escherichia coti is grown in media containing H332p0,; labeled RNA and DNA are differentially extracted by a phenol procedure and purified by treatment with deoxyribonuclease and alkaline hydrolysis, respectively. The RNA and DNA are hydrolyzed with snake venom diesterase, the labeled 5'-nucleotides are separated by chromatography on columns of Dowex-1 anion exchange resin, the nucleotides are phosphorylated to the nucleoside triphosphates by enzyme preparations from E. coli and rabbit muscle, and the triphosphates are purified by chromatography on columns of Dowex 1. Methods are also described for preparing 8*P-labeled ribonucleotide diphosphates. While commercial sources or chemical procedures may be preferable for obtaining single nucleotides labeled with 32p, the biosynthetic procedure is of advantage when a number of labeled nucleotides are required simultaneously, as in determinations of composition or of "nearest-neighbor" frequencies of polynucleotides. Materials
~2P-Orthophosphate (carrier-free) is obtained from Union Carbide Nuclear Company, Bactopeptone and nutrient broth from Difco Laboratories, and crystalline deoxyribonuclease and phosphoenol pyruvate kina~e from Worthington Biochemical Corporation. Snake venom phosphodiesterase, with minimal content of 5'-nucleotide phosphomonoesterase is prepared from Crotalus adamanteus venom (Ross Allen's Reptile Institute, Inc.) by two successive low temperature acetone fractionations or obtained from Worthington Biochemical Corporation. A phosphozTlating enzyme fraction, prepared from E. coli, contains kinases which will convert ribo- and deoxyribonucleotides to the corI D e v e l o p m e n t and use of this procedure was supported by U.S.P.H.S. Grants CA-04464 and CA-06910. Abbreviations used in this article are the same as those used b y the Journal of Biological Chemistry. These procedures were initially based on procedures for the preparation of 8-,p_ deoxynucleotides used at Oak Ridge National Laboratories. See F. J. Bollum, J. Biol. Chem. 235, 2399 (1960).
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NUCLEIC ACID COMPONENTS
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responding nucleoside 5'-triphosphates. E. coli B is grown aerobically on a glucose-inorganic salts medium, harvested before cessation of growth, and .ground with alumina according to Lieberman2 The ground paste is extracted twice with a total of 4 ml of 0.01 M K~HP04-2 mM glutamine5 mM EDTA solution per gram of bacteria, and after a preliminary low speed centrifugation, the supernatant is centrifuged at 100,000 g for 1 hour. The extract is treated with just enough streptomycin sulfate to achieve maximum precipitation and centrifuged at about 25,000 g for 10 minutes. The supernatant is diluted to 12 mg of protein per milliliter and fractionated at 0-2 ° with solid ammonium sulfate at 0-0.21, 0.21-0.30, and 0.30-0.35 g/ml. The precipitate obtained at 0.35 g/ml is dissolved in 0.05 M glycine (pH 8.0)-2 mM glutamine-0.2 mM EDTA to a protein concentration of 8 mg/ml. It contains most of the kinase activity. It has been used without dialysis and is stable for years at --20 °. (The glutamine and EDTA are added to protect other enzymes and may not be essential here.) A glycolyzing enzyme fraction, which catalyzes the phosphorylation of ADP to ATP using potassium phosphoglycerate as a source of high energy phosphate, is prepared from rabbit muscle according to Racker. 4 The final dialyzed enzyme solution is lyophilized and stored at 0-4 ° in a desiccator, where it is stable for years. Method Growth and Harvest o] E. coli. The following medium is used for growth of E. coli B: 7.5 g of Bactopeptone and 12 g of nutrient broth are dissolved in 50 ml of water, adjusted to pH 1 with 4 N HC1, chilled and centrifuged. The precipitate is discarded. The pH is adjusted to 7 with 5 N NaOH and the volume is made to 75 ml. (This mixture may be prepared and stored frozen in quantity.) To 75 ml of the Bactopeptone-nutrient broth concentrate is added 3.7 g of NaC1, 1.5 g of glucose, 3.8 ml of 1 M MgCI2, and 8 ml of 15 M NH40H. The mixture is stirred, the precipitate of MgNH~P0~ is centrifuged out, and the supernatant is added to 1.1 liter of water containing 9.4 g of solid Tris (hydroxymethyl) aminomethane. The pH is adjusted to 7.9-8.0 with 1 N HC1 and 0.9 ml of a solution of inorganic salts is added. This solution, also stored frozen in quantity, contains 2% MgSO4, 0.05% F e S Q . 7 H~O, 0.05% ZnSO~-3 H~O, and 0.005% CuS04.5 H20. The complete medium is autoclaved in a 4-liter heavy-wall erlenmeyer flask fitted with an aeration tube through a cotton and gauze plug; two 20-ml aliquots of the medium are similarly prepared in 125-ml Erlenmeyer flasks. s I. Lieberman, J. Biol. Chem. ,7¢~., 765 (1956). • E. Racker, J. Biol. Chem. 167, 843 (1947).
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Two subcultures are prepared by inoculation of the aliquots with
E. coli B and incubated with aeration overnight at 38 °. One or both of the subcultures is added to the sterile medium along with 50 mC of the unneutralized solution of s2P-orthophosphate5 and antifoam spray, and the E. coli is grown at 38 ° overnight with aeration. The radioactive E. coli is collected by centrifugation for 15 minutes at 7500 rpm in roundbottom 200-ml polycarbonate bottles in a Servall GSA head. T h e pellets are suspended and transferred to a 100 ml glass centrifuge bottle with 80 ml of cold 0.9% NaC1, and centzifuged at 3000 rpm for 15 minutes in the No. 845 head of the International PR-2 centrifuge. Extraction o] Lipids. All extractions are carried out in the 100-ml centrifuge tube with a motor driven paddle stirrer to minimize transfers of radioactive materials. The washed cells are extracted with 80 ml of a 3:1 ethanol-ether solution, containing 2 ml of 1 M ammonium acetate, at 50 ° for 20 minutes with stirring. The suspension is centrifuged and the supernatant is discarded. Extraction of RNA. About 70% of the RNA, containing less than 2% of the DNA, is extracted from the partially lysed cells by a phenol extraction procedure. F o r t y milliliters of water-saturated redistilled phenol and 40 ml of a solution of 0.5% sodium citrate and 0.5% sodium deoxycholate are mixed, heated to 60 °, and added to the cells. The mixture is stirred for 30 minutes at 55 °, then centrifuged at 4000 rpm for 30 minutes at room temperature. The aqueous layer is carefully removed by pipette into another 100-ml tube. The phenol and interphase material are reextracted with 20 ml of citrate-deoxycholate solution by stirring for 10 minutes at 55 ° and centrifugation for 30 minutes. The RNA is precipitated from the combined aqueous phases with 2 volumes of cold ethanol, kept at --20 ° for an hour, centrifuged, and dissolved as described below for treatment with DNase. The phenol and interphase fractions are saved for extraction of DNA. Good separation of R N A and D N A is dependent upon sharp separation of phases and avoidance of contamina£ion of the aqueous phase with interphase material2 ~Safety precautions: In all operations, personnel wear glasses and polyethylene gloves and minimize proximity to the ~'~P. Operations are carried out behind a glass safety shield, mechanical stirring is employed, transfers of solution are made by pipettes fitted with rubber bulbs, all radioactive materials are stored behind lead sheets, and the area and personnel are frequently monitored for contamination by a portable thin-window survey meter. Glassware is reserved for this preparation only. Wastes are stored behind lead for 2 months before disposal. Recovery of RNA. If increased extraction of RNA is desired, with concomitant extraction of DNA, the extraction mixture may include 1 M NaC1 and 0.5% sodium dodecyl sulfate. Two treatments of the mixed nucleates with DNa~ may be necessary.
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~VCL~.IC ACID CO~PON~,NTS
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Extraction o/ DNA. The phenol layer and interphase material are treated with 60 ml of 4:1 ethanol-ether with stirring. T h e proteins and nucleates are centrifuged down and washed once with 40 ml of 0 . 4 N perchloric acid at 0 ° to improve the extractability of D N A . Then 30 ml of cold 2 M potassium acetate and a drop of phenol red are added to the precipitate. The mixture is adjusted to p H 7 with 1 N K 0 H a t 0 °, heated to 100 ° for 15 minutes with stirring, cooled, and centrifuged. The nucleates are precipitated from the s u p e r n a t a n t salt solution by addition of 2 volumes of cold ethanol and recovered by centrifugation. T h e nucleates are dissolved in 20 ml of fresh 0.1 N N a O H and incubated at 3538 ° for 20 hours, to hydrolyze 32P-RNA. The solution is neutralized with HC1 and treated with 2 volumes of ethanol to reprecipitate the D N A . The precipitate is washed twice by suspension and centrifugation a t - - 2 0 ° in 67% ethanol-0.1 M MgC12. The carefully drained precipitate is dissolved in 10 ml of 0.05 M Tris-C1, p H 8.1. Treatment o] RNA Fraction by De,oxyribonuclease. The R N A precipitate is dissolved in 50 ml of w a t e r plus 0.5 ml of 1 M MgC12 and 2.5 ml of 1 M Tris-chloride, p H 7.0. Crystalline deoxyribonuclease (free of ribonuclease), 1.25 mg, is added and the solution is incubated for 2 hours at 38 °. The digest is chilled and made up to 1 M potassium acetate and 0.01 M E D T A . One volume of cold ethanol is added, and after 1 hour at - - 2 0 °, the R N A is centrifuged down. Hydrolysis o] RNA by Diesterase. To the precipitated R N A is added 20 ml of water; 0.2 ml of 1 M Tris-acetate, p H 9.4; 0.02 ml of 1 M MgCl2; 1 ml o f bovine serum albumin, 22 m g / m l ; enough 1 N K 0 H to bring the p H to 9.4; and then 3000 units ~ of snake venom phosphodiesterase free from phosphomonoesterase, s Incubation is for 6-12 hours at 38°; the p H is maintained at p H 9.4 with 1 N K O H . 7One unit of diesterase activity is defined as the hydrolysis of 1 micromole of p-nitrophenyl-5'-phosphothymidine per hour at 38° in an assay mixture of 1.00 ml total volume containing 0.25 ml of 5 mM sodium p-nitrophenyl phosphothymidine (Cal Biochem), 0.05 ml of 1 M Tris buffer to give a final pH of 9.4, 0.01 ml of 1M MgC12 and 0.50 ml of a solution of 1 mg of bovine serum albumin per milliliter. Suitable dilutions of the enzyme are made in the albumin solution. The reaction is stopped after 10-30 minutes by transfer of 0.25-ml aliquots to 1.00 ml of 0.025 M NaOH. The absorbancy is read at 440 millimicrons against a control not containing enzyme. The molar absorbancy of the product, p-nitrophenol, at alkaline pH is 18,600 at 440 millimicrons. Phosphomonoesterase activity is measured at 38° in 1.0 ml of a solution containing 0.40 ml of 0.010 M sodium adenosine 5'-monophosphate, 0.10 ml of 1 M MgCh, and 0.20 ml of 0.25 M Tris-acetate, pH 8.5. Aliquots of 0.30 ml are taken after periods of 1-20 hours and assayed for inorganic phosphate by standard procedures. One unit of activity is defined as the hydrolysis of 1 micromole of AMP per hour. The ratio of phosphomonoesterase units to phosphodiesterase units in the enzyme preparation used for hydrolysis of the RNA should not exceed 0.001.
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t
The extent of hydrolysis is measured by diluting a 10-~1 aliquot with 5.0 ml of a solution of 0.1 mg of crude y e a s t ribonucleate per milliliter, and plating 10-~l aliquots of this dilution on paper disks. The radioactivity of dried disks (total ~2p) is compared with the radioactivity of disks washed twice with cold 5% trichloroacetic acid, twice with ethanol and dried (unhydrolyzed ~*P-RNA). This procedure should indicate 30--50% hydrolysis after a b o u t 2 hours. The reaction is terminated when no further hydrolysis is indicated, usually a b o u t 70-80% of completion. The digest is chilled a t 0 °, made to 0.4 N with concentrated perehloric acid and centrifuged, leaving the labeled 5'-nucleotides in the supernatant. Perchloric acid is removed from the nucleotide solution b y neutralization with 10 N K O H at 0 ° (phenol red is used as an internal indicator). T h e precipitate of KC10, is removed by centrifugation and washed once with 3 ml of cold water. Chromatography o] 5'-Ribonucleotides (el. Hurlbertg). The solution of ribonucleotides is p u t onto a column of Dowex l - X 8 (formate) 200400 mesh, of 15 m m diameter and 150 m m height. T h e column is eluted b y a linear gradient 1° of formic acid (450 ml of water in the mixing flask and 450 ml of 5 M formic acid in the reservoir flask) at a rate of 5 ml per 5 minutes per fraction. Ultraviolet light absorption and radioactivity in the eluate are continuously monitored and recorded. 11 The fractions containing the C M P , A M P , G M P , and U M P peaks are pooled separately into 100-ml r o u n d - b o t t o m flasks, and 0.1 ml of 1 M K O H is added to each flask to neutralize the nucleotides as the formic acid is removed. The formic acid is removed at 30 ° in a continuous-feed r o t a r y evaporator equipped with a mechanical v a c u u m p u m p and a t r a p cooled by D r y Ice 9R. B. Hurlbert, see Vol. III [111]. lo Linear gradient: The linear gradient is obtained by using two identical wide-mouth, fiat-bottom bottles as reservoir and mixer. Both are subjected to the same air pressure and maintain a common liquid level via a siphon connection which extends to the bottoms of both vessels. The siphon includes an intermediate section of Tygon tubing for flexibility; the exit section is bent to clear the magnetic stirring bar, which mixes the eluent, and is constricted at the tip to avoid mixing back of the dilute eluent. The mixed eluent is conducted to the column by a glass tube, bent to clear the stirring bar, and Tygon tubing. Two or more such tubes may be used for simultaneous elution of two or more columns. 11Monitoring of chromatography effluents: Effluent from the column is conducted via size 200-240 polyethylene tubing to a flow cell of 2-mm light path in a spectrophotometer, or other UV monitoring device, set at 260 millimicrons and coupled to a recorder. The ratio-recording Beckman DB spectrophotometer has proved convenient because of the ease of checking absorption at other wavelengths to help identify peaks. The effluent is also conducted past the window of a Geiger tube (with an aluminum shield to moderate the radiation reaching the detector) coupled to ~ ratemeter and recorder. Both recorders have event markers actuated by the fraction collector.
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in acetone. Each monophosphate is dissolved in 6 ml of water as soon as evaporation is complete, transferred to a 4 0 - m l centrifuge tube, and assayed for nucleotide content and 82p. The yield is 20--30 micromoles of each nucleotide. 12 D a t a from a typical preparation are shown in Table I. Preparation o] CMP Free of Deoxy CMP. The ribonucleotides are contaminated with traces of deoxyribonucleotides which do not interfere in most types of work. F o r preparation of C D P used in assay of C D P reductase, this contamination must be removed by chromatography 13 of the C M P fraction. The solution of C M P , p H 3-7, is passed through a column of Dowex 50 ( H ÷) of 10 m m diameter and 130 m m height. T h e TABLE I DATA FROM A TYPICAL PREPARATION: EXTRACTS AND FRACTIONS
Fraction Culture Centrifuged culture supernatant Ethanol-ether extract RNA fraction (after DNAse) DNA fraction (including RNA) RNA 5'-nucleotides after separation
Total cpm~ (millions) 45,500 6,500 2,930 12,000
10,000 7,800
Total RNAb Total DNAb (mg) (rag) --
--
--
--
--
44.0 20.0 --
--
0.26 20.0 --
• Counting is done on aliquots of suitable dilutions of the fraction, plated on paper disks, and counted with a thin-window Geiger counter. These counts are uncorrected for decay. b RNA and DNA were measured colorimetrieally with the orcinol reaction and the diphenylamine reaction, respectively. column is washed with water and eluted with 0.20 M acetic acid a t a rate of 6 ml per 6 minutes per fraction with monitoring. The large ribonucleotide p e a k emerges in a volume of a b o u t 70 ml after about 100 ml of eluent and is followed closely by the deoxynucleotide peak. Phosphorylation o] Ribonucleotides. F o r preparation of the nucleoside triphosphates, the monophosphates are incubated with ATP, a kinase fraction from E. coli, and an A T P regenerating system. The components shown in T a b l e I I are added to the solution of a2p-nucleotide to give a final volume of 10 ml or less. The p H is adjusted to 8.0 before addition of the enzyme fractions. After incubation for 30 minutes at 38 ° , the unlabeled carrier nucleotide is added and the tube is heated to 100 ° for Assay of nucleotides: The concentration and purity of the nucleotides is determined by use of the spectrophotometric data and paper chromatographic methods described by Hurlbert (Vol. I I I [111]) or Furlong (see this volume [39]). P. Reichard, Acta Chem. Scan& 12, 2048 (1958).
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2 minutes. All tubes are stored frozen unless used immediately and are centrifuged before chromatography. If both the diphosphate and triphosphate of cytidine, uridine, or guanosine are desired, the amounts of ATP and phosphoglycerate are reduced to 10 micromoles each during the incubation, and 2 micromoles of the appropriate nucleoside diphosphate plus 30 micromoles of ADP are added 2 minutes prior to heating of the reaction mixture. About onehalf of the product is the nucleoside diphosphate under these conditions.
Chromatography o] the Ribonucleoside Triphosphates ( c]. Hurlbertg). The phosphorylation mixtures are each put onto a separate column of Dowex-1 (formate) 10 mm in diameter and 250 mm in height. The CTP, TABLE II CONDITIONS FOR PHOSPHORYLATION OF RIBONUCLEOSIDE 5/-PHosPHATES
3*P-Nucleotidephosphorylated Compound Incubation component ATP (~moles) Tris-acetate, pH 8.0 (~moles) MgCl2 (~moles) Kophosphoglycerate(~moles) E. coli ldnase fraction (ml) Muscle glycolysisfraction (mg) Added after incubation Carrier nucleotide Amount added (~moles)
AMP
GMP
UMP
CMP
25 375 300 200 1.0 10
100 375 300 100 1.0 10
100 375 300 100 1.0 10
100 375 300 100 1.0 10
---
GTP 20
UTP 20
CTP 20
ATP, and UTP preparations are chromatographed by a linear gradient TM (250 ml of water in the mixer and 250 ml of 4 M formic acid-l.2 M ammonium formate in the reservoir) at the rate of 7 ml per fraction per 5 minutes. The chromatography of G T P requires 250 ml of 4 M formic acid-l.8 M ammonium formate in the reservoir. The fractions containing a nueleoside triphosphate are pooled and passed through a column of Dowex 50 (H ÷) (freshly washed with 4 N HC1 and water) at 0 ° to remove ammonium ions. The columns are 28 mm in diameter and 50 mm in height for ATP and CTP, 28 mm X 70 mm for U T P and GTP, and 20 X 40 mm for CDP. Conversion of the resin to the ammonium ion form may be observed as a band of slightly darker color. Each column is washed with 40-60 ml of cold water. The total effluents are collected, 0.10 ml of 1 M K 0 H is added to each and each is evaporated at low temperature and pressure in a small round-bottom flask in the rotary evaporator until just dry. The ATP fraction sometimes contains a non-
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volatile component which requires more than the above amount of alkali for neutralization. Each triphosphate is dissolved in 6 ml of water, neutralized with KOH, standardized, and stored frozen. D a t a from a typical preparation are shown in Table III. The diphosphates are eluted before the triphosphates as previously described 9 and are recovered by comparable procedures. Hydrolysis o] DNA by Diesterase. The following solutions are added to the 10 ml of DNA: 0.2 ml of 1 M MgCl2, 0.5 ml of 10 mg/ml bovine serum albumin, 0.5 ml of 1.0 gm/ml pancreatic DNase, and 0.5 ml of TABLE III DATA FROM A TYPICAL PREPARATION: NUCLEOTIDES
3~P-Nucleotide
Total cpm (millions)
Total micromolesa
Specific activity* (cpm/ttmole, millions)
CTP ATP GTP UTP dAMP dCMP dGMP dTMP dATP dCTP dGTP TTP
645 627 756 727 204 150 130 159 32.8 24.9 24.1 25.4
22.0 25.0 27.6 24.2 4.2 4.7 3.6 4.9 2.59 2.46 2.38 2.51
21.8 25.1 27.5 30.2 48.5 32.0 36.0 32.5 12.6 10.1 10.1 10.1
a Amounts of nucleotides are quantitated by spectrophotometry (see text footnote 12). b Radioactivity is determined by plating suitable dilutions on filter paper disks and
counting these with a thin-window Geiger counter. 5.0 mg/ml commercial snake venom diesterase. The solution is incubated at 37 ° for 4 hours with occasional stirring and then heated at 100 ° for 5 minutes. The coagulated proteins are removed by centrifugation and washed twice with 2 ml of distilled water. The combined supernatant and wash are pooled, adjusted to about p H 10 with concentrated N H 4 0 H and stored frozen for subsequent chromatography. Chromatography o] 5"Deoxyribonucleotides. The chromatography and desalting of the deoxynucleotides differs from t h a t described for the ribonueleotides to avoid the use of concentrated acid solutions which would hydrolyze the purine deoxyribonucleotides. These techniques are modified from those used by Cohn and Bollum. 14 The diesterase digest 14W. E. Cohn and F. J. Bollum, Biochim. Biophys. Acta 48, 588 (1961).
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containing nucleotides derived from DNA is passed through a 10 X 100 mm column of Dowex l-X8 (formate, 200-400 mesh), and the column is washed with water. The effluents are monitored11 for both light absorption and ~2p. The dCMP is eluted by 50 ml of 0.05 M formic acid at a flow rate of 2 ml per minute. The dAMP is eluted by 100 ml of 0.3 M formic acid. The dCMP and dAMP are immediately adjusted to pH 10.0 with NH4OH and diluted 1:3 with water. They are recaptured on columns of Dowex 1 (formate) of 10 mm diameter and 20 mm height. These short columns and the main column are washed with water, and the resin is converted to the carbonate form by treatment with 0.1 M Na~C0s, 20 ml for the short columns and 100 ml for the long column. Excess salt is washed from the columns with water. The dCMP and dAMP are eluted from the short columns by 10 ml of 0.6 M ammonium bicarbonate. The T M P and dGMP are sequentially eluted from the long column by use of 50 ml of 0.2 M ammonium bicarbonate followed by 150 ml of 0.75 M ammonium bicarbonate. The nucleotides dCMP, dAMP, TMP, and dGMP are desalted by evaporation from a 40 ° water bath in a rotary evaporator evacuated by a water aspirator. Each monophosphate is redissolved in 3-5 ml of H20. Phosphorylation o] Deoxyribonucleotides. The deoxyribonucleoside triphosphates are prepared in a reaction mixture with the following components; 0.50 ml of 1.0M Tris-chloride, pH 8.1; 0.25 ml of 0.2M MgC12; 0.10 ml of 2.5M KC1; 0.10 ml of 0.1M mercaptoethanol; 0.10 ml of sodium EDTA, pH 8.1; 0.10 ml of 0.25 M phosphoenol pyruvate; 0.010 ml of phosphoenol pyruvate kinase solution, 10 mg/ml; 3.30 ml of the 3~P-nucleotide solution, 1.5-3.0 mM and 0.50 ml of the E. coli kinase fraction. For the phosphorylation of dAMP, 0.005 ml of 0.25 M ATP is added; for the other nucleotides, 0.020 ml of 0.25 M ATP is used. The total reaction volume is 5.0 ml. The reaction solution is incubated for 2 hours at 35 °, heated to 95 ° for 3 minutes and centrifuged to remove coagulated proteins. Chromatography o] Deoxyribonucleoside Triphosphates. The supernatants containing the 32P-dATP and the ~2p-dCTP are loaded on separate 10 mm X 200 mm Dowex 1 (carbonate) columns and eluted with a linear gradient, produced by 150 ml of water in the mixing flask and 150 ml of 1.0M ammonium bicarbonate in the reservoir, at a flow rate of 2 ml per minute. The dCTP appears approximately halfway through the gradient and the dATP is eluted during the final quarter of the gradient. Supernatant fluids containing ~2P-TTP and ~'-'P-dGTP are loaded onto separate 10 X 200 mm columns of Dowex 1 (formate) and eluted with a linear gradient produced by 50 ml of water in the mixing flask and 50 ml of 4 M formic acid-1 M ammonium formate in the
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reservoir. ATP is eluted near the end of this gradient and recognized by the high absorbance and low radioactivity. The columns are washed wi~h water, changed to the carbonate form with 100 ml of 0.2 M Na2C03 and washed with water to remove excess salt. Then 50-75 ml of 0.75 M ammonium bicarbonate is used to elute T T P and 75-100 ml of 1.5 M ammonium bicarbonate is used to elute dGTP. The fractions containing the triphosphates are evaporated and desalted in va cu o in a rotary evaporator at 40 °, and taken up in 3-5 ml of water. The nucleotides are assayed12 and stored frozen. Data from a typical preparation are given in Table III.
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[24b] B i o s y n t h e t i c P r e p a r a t i o n of 3 2 p - L a b e l e d N u c l e o s i d e 5'-Phosphates and Derivatives 1
By ROBERT B.
HURLBERT and N. BURR FURLONG
Introduction A method s is described for the biosynthetic and enzymatic preparation of CTP, ATP, GTP, UTP, CDP, dATP, dCTP, dGTP, and TTP labeled with 32p in the alpha position. Escherichia coti is grown in media containing H332p0,; labeled RNA and DNA are differentially extracted by a phenol procedure and purified by treatment with deoxyribonuclease and alkaline hydrolysis, respectively. The RNA and DNA are hydrolyzed with snake venom diesterase, the labeled 5'-nucleotides are separated by chromatography on columns of Dowex-1 anion exchange resin, the nucleotides are phosphorylated to the nucleoside triphosphates by enzyme preparations from E. coli and rabbit muscle, and the triphosphates are purified by chromatography on columns of Dowex 1. Methods are also described for preparing 8*P-labeled ribonucleotide diphosphates. While commercial sources or chemical procedures may be preferable for obtaining single nucleotides labeled with 32p, the biosynthetic procedure is of advantage when a number of labeled nucleotides are required simultaneously, as in determinations of composition or of "nearest-neighbor" frequencies of polynucleotides. Materials
~2P-Orthophosphate (carrier-free) is obtained from Union Carbide Nuclear Company, Bactopeptone and nutrient broth from Difco Laboratories, and crystalline deoxyribonuclease and phosphoenol pyruvate kina~e from Worthington Biochemical Corporation. Snake venom phosphodiesterase, with minimal content of 5'-nucleotide phosphomonoesterase is prepared from Crotalus adamanteus venom (Ross Allen's Reptile Institute, Inc.) by two successive low temperature acetone fractionations or obtained from Worthington Biochemical Corporation. A phosphozTlating enzyme fraction, prepared from E. coli, contains kinases which will convert ribo- and deoxyribonucleotides to the corI D e v e l o p m e n t and use of this procedure was supported by U.S.P.H.S. Grants CA-04464 and CA-06910. Abbreviations used in this article are the same as those used b y the Journal of Biological Chemistry. These procedures were initially based on procedures for the preparation of 8-,p_ deoxynucleotides used at Oak Ridge National Laboratories. See F. J. Bollum, J. Biol. Chem. 235, 2399 (1960).
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responding nucleoside 5'-triphosphates. E. coli B is grown aerobically on a glucose-inorganic salts medium, harvested before cessation of growth, and .ground with alumina according to Lieberman2 The ground paste is extracted twice with a total of 4 ml of 0.01 M K~HP04-2 mM glutamine5 mM EDTA solution per gram of bacteria, and after a preliminary low speed centrifugation, the supernatant is centrifuged at 100,000 g for 1 hour. The extract is treated with just enough streptomycin sulfate to achieve maximum precipitation and centrifuged at about 25,000 g for 10 minutes. The supernatant is diluted to 12 mg of protein per milliliter and fractionated at 0-2 ° with solid ammonium sulfate at 0-0.21, 0.21-0.30, and 0.30-0.35 g/ml. The precipitate obtained at 0.35 g/ml is dissolved in 0.05 M glycine (pH 8.0)-2 mM glutamine-0.2 mM EDTA to a protein concentration of 8 mg/ml. It contains most of the kinase activity. It has been used without dialysis and is stable for years at --20 °. (The glutamine and EDTA are added to protect other enzymes and may not be essential here.) A glycolyzing enzyme fraction, which catalyzes the phosphorylation of ADP to ATP using potassium phosphoglycerate as a source of high energy phosphate, is prepared from rabbit muscle according to Racker. 4 The final dialyzed enzyme solution is lyophilized and stored at 0-4 ° in a desiccator, where it is stable for years. Method Growth and Harvest o] E. coli. The following medium is used for growth of E. coli B: 7.5 g of Bactopeptone and 12 g of nutrient broth are dissolved in 50 ml of water, adjusted to pH 1 with 4 N HC1, chilled and centrifuged. The precipitate is discarded. The pH is adjusted to 7 with 5 N NaOH and the volume is made to 75 ml. (This mixture may be prepared and stored frozen in quantity.) To 75 ml of the Bactopeptone-nutrient broth concentrate is added 3.7 g of NaC1, 1.5 g of glucose, 3.8 ml of 1 M MgCI2, and 8 ml of 15 M NH40H. The mixture is stirred, the precipitate of MgNH~P0~ is centrifuged out, and the supernatant is added to 1.1 liter of water containing 9.4 g of solid Tris (hydroxymethyl) aminomethane. The pH is adjusted to 7.9-8.0 with 1 N HC1 and 0.9 ml of a solution of inorganic salts is added. This solution, also stored frozen in quantity, contains 2% MgSO4, 0.05% F e S Q . 7 H~O, 0.05% ZnSO~-3 H~O, and 0.005% CuS04.5 H20. The complete medium is autoclaved in a 4-liter heavy-wall erlenmeyer flask fitted with an aeration tube through a cotton and gauze plug; two 20-ml aliquots of the medium are similarly prepared in 125-ml Erlenmeyer flasks. s I. Lieberman, J. Biol. Chem. ,7¢~., 765 (1956). • E. Racker, J. Biol. Chem. 167, 843 (1947).
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195
Two subcultures are prepared by inoculation of the aliquots with
E. coli B and incubated with aeration overnight at 38 °. One or both of the subcultures is added to the sterile medium along with 50 mC of the unneutralized solution of s2P-orthophosphate5 and antifoam spray, and the E. coli is grown at 38 ° overnight with aeration. The radioactive E. coli is collected by centrifugation for 15 minutes at 7500 rpm in roundbottom 200-ml polycarbonate bottles in a Servall GSA head. T h e pellets are suspended and transferred to a 100 ml glass centrifuge bottle with 80 ml of cold 0.9% NaC1, and centzifuged at 3000 rpm for 15 minutes in the No. 845 head of the International PR-2 centrifuge. Extraction o] Lipids. All extractions are carried out in the 100-ml centrifuge tube with a motor driven paddle stirrer to minimize transfers of radioactive materials. The washed cells are extracted with 80 ml of a 3:1 ethanol-ether solution, containing 2 ml of 1 M ammonium acetate, at 50 ° for 20 minutes with stirring. The suspension is centrifuged and the supernatant is discarded. Extraction of RNA. About 70% of the RNA, containing less than 2% of the DNA, is extracted from the partially lysed cells by a phenol extraction procedure. F o r t y milliliters of water-saturated redistilled phenol and 40 ml of a solution of 0.5% sodium citrate and 0.5% sodium deoxycholate are mixed, heated to 60 °, and added to the cells. The mixture is stirred for 30 minutes at 55 °, then centrifuged at 4000 rpm for 30 minutes at room temperature. The aqueous layer is carefully removed by pipette into another 100-ml tube. The phenol and interphase material are reextracted with 20 ml of citrate-deoxycholate solution by stirring for 10 minutes at 55 ° and centrifugation for 30 minutes. The RNA is precipitated from the combined aqueous phases with 2 volumes of cold ethanol, kept at --20 ° for an hour, centrifuged, and dissolved as described below for treatment with DNase. The phenol and interphase fractions are saved for extraction of DNA. Good separation of R N A and D N A is dependent upon sharp separation of phases and avoidance of contamina£ion of the aqueous phase with interphase material2 ~Safety precautions: In all operations, personnel wear glasses and polyethylene gloves and minimize proximity to the ~'~P. Operations are carried out behind a glass safety shield, mechanical stirring is employed, transfers of solution are made by pipettes fitted with rubber bulbs, all radioactive materials are stored behind lead sheets, and the area and personnel are frequently monitored for contamination by a portable thin-window survey meter. Glassware is reserved for this preparation only. Wastes are stored behind lead for 2 months before disposal. Recovery of RNA. If increased extraction of RNA is desired, with concomitant extraction of DNA, the extraction mixture may include 1 M NaC1 and 0.5% sodium dodecyl sulfate. Two treatments of the mixed nucleates with DNa~ may be necessary.
196
~VCL~.IC ACID CO~PON~,NTS
[24b]
Extraction o/ DNA. The phenol layer and interphase material are treated with 60 ml of 4:1 ethanol-ether with stirring. T h e proteins and nucleates are centrifuged down and washed once with 40 ml of 0 . 4 N perchloric acid at 0 ° to improve the extractability of D N A . Then 30 ml of cold 2 M potassium acetate and a drop of phenol red are added to the precipitate. The mixture is adjusted to p H 7 with 1 N K 0 H a t 0 °, heated to 100 ° for 15 minutes with stirring, cooled, and centrifuged. The nucleates are precipitated from the s u p e r n a t a n t salt solution by addition of 2 volumes of cold ethanol and recovered by centrifugation. T h e nucleates are dissolved in 20 ml of fresh 0.1 N N a O H and incubated at 3538 ° for 20 hours, to hydrolyze 32P-RNA. The solution is neutralized with HC1 and treated with 2 volumes of ethanol to reprecipitate the D N A . The precipitate is washed twice by suspension and centrifugation a t - - 2 0 ° in 67% ethanol-0.1 M MgC12. The carefully drained precipitate is dissolved in 10 ml of 0.05 M Tris-C1, p H 8.1. Treatment o] RNA Fraction by De,oxyribonuclease. The R N A precipitate is dissolved in 50 ml of w a t e r plus 0.5 ml of 1 M MgC12 and 2.5 ml of 1 M Tris-chloride, p H 7.0. Crystalline deoxyribonuclease (free of ribonuclease), 1.25 mg, is added and the solution is incubated for 2 hours at 38 °. The digest is chilled and made up to 1 M potassium acetate and 0.01 M E D T A . One volume of cold ethanol is added, and after 1 hour at - - 2 0 °, the R N A is centrifuged down. Hydrolysis o] RNA by Diesterase. To the precipitated R N A is added 20 ml of water; 0.2 ml of 1 M Tris-acetate, p H 9.4; 0.02 ml of 1 M MgCl2; 1 ml o f bovine serum albumin, 22 m g / m l ; enough 1 N K 0 H to bring the p H to 9.4; and then 3000 units ~ of snake venom phosphodiesterase free from phosphomonoesterase, s Incubation is for 6-12 hours at 38°; the p H is maintained at p H 9.4 with 1 N K O H . 7One unit of diesterase activity is defined as the hydrolysis of 1 micromole of p-nitrophenyl-5'-phosphothymidine per hour at 38° in an assay mixture of 1.00 ml total volume containing 0.25 ml of 5 mM sodium p-nitrophenyl phosphothymidine (Cal Biochem), 0.05 ml of 1 M Tris buffer to give a final pH of 9.4, 0.01 ml of 1M MgC12 and 0.50 ml of a solution of 1 mg of bovine serum albumin per milliliter. Suitable dilutions of the enzyme are made in the albumin solution. The reaction is stopped after 10-30 minutes by transfer of 0.25-ml aliquots to 1.00 ml of 0.025 M NaOH. The absorbancy is read at 440 millimicrons against a control not containing enzyme. The molar absorbancy of the product, p-nitrophenol, at alkaline pH is 18,600 at 440 millimicrons. Phosphomonoesterase activity is measured at 38° in 1.0 ml of a solution containing 0.40 ml of 0.010 M sodium adenosine 5'-monophosphate, 0.10 ml of 1 M MgCh, and 0.20 ml of 0.25 M Tris-acetate, pH 8.5. Aliquots of 0.30 ml are taken after periods of 1-20 hours and assayed for inorganic phosphate by standard procedures. One unit of activity is defined as the hydrolysis of 1 micromole of AMP per hour. The ratio of phosphomonoesterase units to phosphodiesterase units in the enzyme preparation used for hydrolysis of the RNA should not exceed 0.001.
[24b]
32P-LABELED NUCLEOSIDE TRIPHOSPHATES--BIOSYNTHETIC
197
t
The extent of hydrolysis is measured by diluting a 10-~1 aliquot with 5.0 ml of a solution of 0.1 mg of crude y e a s t ribonucleate per milliliter, and plating 10-~l aliquots of this dilution on paper disks. The radioactivity of dried disks (total ~2p) is compared with the radioactivity of disks washed twice with cold 5% trichloroacetic acid, twice with ethanol and dried (unhydrolyzed ~*P-RNA). This procedure should indicate 30--50% hydrolysis after a b o u t 2 hours. The reaction is terminated when no further hydrolysis is indicated, usually a b o u t 70-80% of completion. The digest is chilled a t 0 °, made to 0.4 N with concentrated perehloric acid and centrifuged, leaving the labeled 5'-nucleotides in the supernatant. Perchloric acid is removed from the nucleotide solution b y neutralization with 10 N K O H at 0 ° (phenol red is used as an internal indicator). T h e precipitate of KC10, is removed by centrifugation and washed once with 3 ml of cold water. Chromatography o] 5'-Ribonucleotides (el. Hurlbertg). The solution of ribonucleotides is p u t onto a column of Dowex l - X 8 (formate) 200400 mesh, of 15 m m diameter and 150 m m height. T h e column is eluted b y a linear gradient 1° of formic acid (450 ml of water in the mixing flask and 450 ml of 5 M formic acid in the reservoir flask) at a rate of 5 ml per 5 minutes per fraction. Ultraviolet light absorption and radioactivity in the eluate are continuously monitored and recorded. 11 The fractions containing the C M P , A M P , G M P , and U M P peaks are pooled separately into 100-ml r o u n d - b o t t o m flasks, and 0.1 ml of 1 M K O H is added to each flask to neutralize the nucleotides as the formic acid is removed. The formic acid is removed at 30 ° in a continuous-feed r o t a r y evaporator equipped with a mechanical v a c u u m p u m p and a t r a p cooled by D r y Ice 9R. B. Hurlbert, see Vol. III [111]. lo Linear gradient: The linear gradient is obtained by using two identical wide-mouth, fiat-bottom bottles as reservoir and mixer. Both are subjected to the same air pressure and maintain a common liquid level via a siphon connection which extends to the bottoms of both vessels. The siphon includes an intermediate section of Tygon tubing for flexibility; the exit section is bent to clear the magnetic stirring bar, which mixes the eluent, and is constricted at the tip to avoid mixing back of the dilute eluent. The mixed eluent is conducted to the column by a glass tube, bent to clear the stirring bar, and Tygon tubing. Two or more such tubes may be used for simultaneous elution of two or more columns. 11Monitoring of chromatography effluents: Effluent from the column is conducted via size 200-240 polyethylene tubing to a flow cell of 2-mm light path in a spectrophotometer, or other UV monitoring device, set at 260 millimicrons and coupled to a recorder. The ratio-recording Beckman DB spectrophotometer has proved convenient because of the ease of checking absorption at other wavelengths to help identify peaks. The effluent is also conducted past the window of a Geiger tube (with an aluminum shield to moderate the radiation reaching the detector) coupled to ~ ratemeter and recorder. Both recorders have event markers actuated by the fraction collector.
198
[24b]
I~UCLEIC ACID COMPONENTS
in acetone. Each monophosphate is dissolved in 6 ml of water as soon as evaporation is complete, transferred to a 4 0 - m l centrifuge tube, and assayed for nucleotide content and 82p. The yield is 20--30 micromoles of each nucleotide. 12 D a t a from a typical preparation are shown in Table I. Preparation o] CMP Free of Deoxy CMP. The ribonucleotides are contaminated with traces of deoxyribonucleotides which do not interfere in most types of work. F o r preparation of C D P used in assay of C D P reductase, this contamination must be removed by chromatography 13 of the C M P fraction. The solution of C M P , p H 3-7, is passed through a column of Dowex 50 ( H ÷) of 10 m m diameter and 130 m m height. T h e TABLE I DATA FROM A TYPICAL PREPARATION: EXTRACTS AND FRACTIONS
Fraction Culture Centrifuged culture supernatant Ethanol-ether extract RNA fraction (after DNAse) DNA fraction (including RNA) RNA 5'-nucleotides after separation
Total cpm~ (millions) 45,500 6,500 2,930 12,000
10,000 7,800
Total RNAb Total DNAb (mg) (rag) --
--
--
--
--
44.0 20.0 --
--
0.26 20.0 --
• Counting is done on aliquots of suitable dilutions of the fraction, plated on paper disks, and counted with a thin-window Geiger counter. These counts are uncorrected for decay. b RNA and DNA were measured colorimetrieally with the orcinol reaction and the diphenylamine reaction, respectively. column is washed with water and eluted with 0.20 M acetic acid a t a rate of 6 ml per 6 minutes per fraction with monitoring. The large ribonucleotide p e a k emerges in a volume of a b o u t 70 ml after about 100 ml of eluent and is followed closely by the deoxynucleotide peak. Phosphorylation o] Ribonucleotides. F o r preparation of the nucleoside triphosphates, the monophosphates are incubated with ATP, a kinase fraction from E. coli, and an A T P regenerating system. The components shown in T a b l e I I are added to the solution of a2p-nucleotide to give a final volume of 10 ml or less. The p H is adjusted to 8.0 before addition of the enzyme fractions. After incubation for 30 minutes at 38 ° , the unlabeled carrier nucleotide is added and the tube is heated to 100 ° for Assay of nucleotides: The concentration and purity of the nucleotides is determined by use of the spectrophotometric data and paper chromatographic methods described by Hurlbert (Vol. I I I [111]) or Furlong (see this volume [39]). P. Reichard, Acta Chem. Scan& 12, 2048 (1958).
[24b]
32p-LABELED NUCLEOSIDE TRIPHOSPHATES--BIOSYNTHETIC
199
2 minutes. All tubes are stored frozen unless used immediately and are centrifuged before chromatography. If both the diphosphate and triphosphate of cytidine, uridine, or guanosine are desired, the amounts of ATP and phosphoglycerate are reduced to 10 micromoles each during the incubation, and 2 micromoles of the appropriate nucleoside diphosphate plus 30 micromoles of ADP are added 2 minutes prior to heating of the reaction mixture. About onehalf of the product is the nucleoside diphosphate under these conditions.
Chromatography o] the Ribonucleoside Triphosphates ( c]. Hurlbertg). The phosphorylation mixtures are each put onto a separate column of Dowex-1 (formate) 10 mm in diameter and 250 mm in height. The CTP, TABLE II CONDITIONS FOR PHOSPHORYLATION OF RIBONUCLEOSIDE 5/-PHosPHATES
3*P-Nucleotidephosphorylated Compound Incubation component ATP (~moles) Tris-acetate, pH 8.0 (~moles) MgCl2 (~moles) Kophosphoglycerate(~moles) E. coli ldnase fraction (ml) Muscle glycolysisfraction (mg) Added after incubation Carrier nucleotide Amount added (~moles)
AMP
GMP
UMP
CMP
25 375 300 200 1.0 10
100 375 300 100 1.0 10
100 375 300 100 1.0 10
100 375 300 100 1.0 10
---
GTP 20
UTP 20
CTP 20
ATP, and UTP preparations are chromatographed by a linear gradient TM (250 ml of water in the mixer and 250 ml of 4 M formic acid-l.2 M ammonium formate in the reservoir) at the rate of 7 ml per fraction per 5 minutes. The chromatography of G T P requires 250 ml of 4 M formic acid-l.8 M ammonium formate in the reservoir. The fractions containing a nueleoside triphosphate are pooled and passed through a column of Dowex 50 (H ÷) (freshly washed with 4 N HC1 and water) at 0 ° to remove ammonium ions. The columns are 28 mm in diameter and 50 mm in height for ATP and CTP, 28 mm X 70 mm for U T P and GTP, and 20 X 40 mm for CDP. Conversion of the resin to the ammonium ion form may be observed as a band of slightly darker color. Each column is washed with 40-60 ml of cold water. The total effluents are collected, 0.10 ml of 1 M K 0 H is added to each and each is evaporated at low temperature and pressure in a small round-bottom flask in the rotary evaporator until just dry. The ATP fraction sometimes contains a non-
200
NUCLEIC ACID COMPONENTS
[24b]
volatile component which requires more than the above amount of alkali for neutralization. Each triphosphate is dissolved in 6 ml of water, neutralized with KOH, standardized, and stored frozen. D a t a from a typical preparation are shown in Table III. The diphosphates are eluted before the triphosphates as previously described 9 and are recovered by comparable procedures. Hydrolysis o] DNA by Diesterase. The following solutions are added to the 10 ml of DNA: 0.2 ml of 1 M MgCl2, 0.5 ml of 10 mg/ml bovine serum albumin, 0.5 ml of 1.0 gm/ml pancreatic DNase, and 0.5 ml of TABLE III DATA FROM A TYPICAL PREPARATION: NUCLEOTIDES
3~P-Nucleotide
Total cpm (millions)
Total micromolesa
Specific activity* (cpm/ttmole, millions)
CTP ATP GTP UTP dAMP dCMP dGMP dTMP dATP dCTP dGTP TTP
645 627 756 727 204 150 130 159 32.8 24.9 24.1 25.4
22.0 25.0 27.6 24.2 4.2 4.7 3.6 4.9 2.59 2.46 2.38 2.51
21.8 25.1 27.5 30.2 48.5 32.0 36.0 32.5 12.6 10.1 10.1 10.1
a Amounts of nucleotides are quantitated by spectrophotometry (see text footnote 12). b Radioactivity is determined by plating suitable dilutions on filter paper disks and
counting these with a thin-window Geiger counter. 5.0 mg/ml commercial snake venom diesterase. The solution is incubated at 37 ° for 4 hours with occasional stirring and then heated at 100 ° for 5 minutes. The coagulated proteins are removed by centrifugation and washed twice with 2 ml of distilled water. The combined supernatant and wash are pooled, adjusted to about p H 10 with concentrated N H 4 0 H and stored frozen for subsequent chromatography. Chromatography o] 5"Deoxyribonucleotides. The chromatography and desalting of the deoxynucleotides differs from t h a t described for the ribonueleotides to avoid the use of concentrated acid solutions which would hydrolyze the purine deoxyribonucleotides. These techniques are modified from those used by Cohn and Bollum. 14 The diesterase digest 14W. E. Cohn and F. J. Bollum, Biochim. Biophys. Acta 48, 588 (1961).
[24b]
32P-LABELED NUCLEOSIDE TRIPHOSPHATES--BIOSYNTHETIC
201
containing nucleotides derived from DNA is passed through a 10 X 100 mm column of Dowex l-X8 (formate, 200-400 mesh), and the column is washed with water. The effluents are monitored11 for both light absorption and ~2p. The dCMP is eluted by 50 ml of 0.05 M formic acid at a flow rate of 2 ml per minute. The dAMP is eluted by 100 ml of 0.3 M formic acid. The dCMP and dAMP are immediately adjusted to pH 10.0 with NH4OH and diluted 1:3 with water. They are recaptured on columns of Dowex 1 (formate) of 10 mm diameter and 20 mm height. These short columns and the main column are washed with water, and the resin is converted to the carbonate form by treatment with 0.1 M Na~C0s, 20 ml for the short columns and 100 ml for the long column. Excess salt is washed from the columns with water. The dCMP and dAMP are eluted from the short columns by 10 ml of 0.6 M ammonium bicarbonate. The T M P and dGMP are sequentially eluted from the long column by use of 50 ml of 0.2 M ammonium bicarbonate followed by 150 ml of 0.75 M ammonium bicarbonate. The nucleotides dCMP, dAMP, TMP, and dGMP are desalted by evaporation from a 40 ° water bath in a rotary evaporator evacuated by a water aspirator. Each monophosphate is redissolved in 3-5 ml of H20. Phosphorylation o] Deoxyribonucleotides. The deoxyribonucleoside triphosphates are prepared in a reaction mixture with the following components; 0.50 ml of 1.0M Tris-chloride, pH 8.1; 0.25 ml of 0.2M MgC12; 0.10 ml of 2.5M KC1; 0.10 ml of 0.1M mercaptoethanol; 0.10 ml of sodium EDTA, pH 8.1; 0.10 ml of 0.25 M phosphoenol pyruvate; 0.010 ml of phosphoenol pyruvate kinase solution, 10 mg/ml; 3.30 ml of the 3~P-nucleotide solution, 1.5-3.0 mM and 0.50 ml of the E. coli kinase fraction. For the phosphorylation of dAMP, 0.005 ml of 0.25 M ATP is added; for the other nucleotides, 0.020 ml of 0.25 M ATP is used. The total reaction volume is 5.0 ml. The reaction solution is incubated for 2 hours at 35 °, heated to 95 ° for 3 minutes and centrifuged to remove coagulated proteins. Chromatography o] Deoxyribonucleoside Triphosphates. The supernatants containing the 32P-dATP and the ~2p-dCTP are loaded on separate 10 mm X 200 mm Dowex 1 (carbonate) columns and eluted with a linear gradient, produced by 150 ml of water in the mixing flask and 150 ml of 1.0M ammonium bicarbonate in the reservoir, at a flow rate of 2 ml per minute. The dCTP appears approximately halfway through the gradient and the dATP is eluted during the final quarter of the gradient. Supernatant fluids containing ~2P-TTP and ~'-'P-dGTP are loaded onto separate 10 X 200 mm columns of Dowex 1 (formate) and eluted with a linear gradient produced by 50 ml of water in the mixing flask and 50 ml of 4 M formic acid-1 M ammonium formate in the
202
NUCLEIC ACID COMPONENTS
[24b]
reservoir. ATP is eluted near the end of this gradient and recognized by the high absorbance and low radioactivity. The columns are washed wi~h water, changed to the carbonate form with 100 ml of 0.2 M Na2C03 and washed with water to remove excess salt. Then 50-75 ml of 0.75 M ammonium bicarbonate is used to elute T T P and 75-100 ml of 1.5 M ammonium bicarbonate is used to elute dGTP. The fractions containing the triphosphates are evaporated and desalted in va cu o in a rotary evaporator at 40 °, and taken up in 3-5 ml of water. The nucleotides are assayed12 and stored frozen. Data from a typical preparation are given in Table III.