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[39] Preparation and assay of acetyl phosphate
228
CARBOHYDRATES
[39]
Bandurski and Axelrod 8 described conditions for the identification of phosphopyruvic acid by two-dimensional paper chromatography. Utter and Kurahashi 9 found that phosphopyruvic acid may be purified efficiently by a passage through a column of Dowex 1 resin. They obtained by this step the acid prepared from the first barium precipitate of the Baer-Fischer synthesis 6 in 50 % yield and in a state of 90% purity. Stability. Silver barium phosphopyruvate is stable for years at room temperature in brown glass containers. A slight discoloration after long storage may be disregarded if the salt is being used for the preparation of phosphopyruvic acid. Drying Conditions. 1 At 78 °, silver barium phosphopyruvate loses, per mole, between 0.9 and 1 mole of its 2 moles of crystal water within 1 hour in high vacuum over P2Os. Drying at 100 ° in a high vacuum results in discoloration and larger loss of weight. s R. S. Bandurski and B. Axelrod, J. Biol. Chem. 193, 405 (1951). 9 M. F. Utter and K. Kurahashi, J. Biol Chem. 207, 821 (1954).
[39] Preparation and Assay of Acetyl Phosphate B y E . R. STADTMAN
Preparation of Acetyl Phosphate (Acetyl~-~P) by Reaction of Orthophosphate with Isopropenyl Acetate (Procedure A) Principle. This method, which has been previously described, ~ is based on the fact that orthophosphoric acid is readily acetylated by isopropenyl acetate to give monoacetyl dihydrogen phosphate. The product is isolated as the dilithium salt by fractional crystallization from dilute alcoholic solution. Reagents 85% sirupy phosphoric acid. Isopropenyl acetate (freshly distilled). Concentrated sulfuric acid. Lithium hydroxide, 4 N. Procedure. Two hundred milliliters of isopropenyl acetate in a 1-1. flask is cooled in an ice bath. Then, with stirring, 25 ml. of 85 % sirupy phosphoric acid is added dropwise, and finally 1.0 ml. of concentrated sulfuric acid is added as catalyst. The reaction mixture is placed in a water bath at 30° and is stirred constantly to prevent localizing heating. l E. R. Stadtman and F. Lipmann, J. Biol. Chem. 185, 549 (1950).
[39]
PREPARATION AND ASSAY OF ACETYL PHOSPHATE
229
After 30 minutes, or when the reaction mixture becomes pale yellow in color, it is cooled in an ice bath and 100 g. of ice is added. The mixture is neutralized to pH 5.0 with cold 4 N lithium hydroxide (about 300 ml.). During neutralization, the mixture may separate into two phases, since the isopropenyl acetate which is present in excess is relatively insoluble in water. It is therefore necessary to shake the mixture vigorously in an ice bath during the addition of lithium hydroxide so as to extract the acetyl phosphoric acid from the organic phase. The neutralization should be carried out as rapidly as possible without, however, allowing the temperature to rise unduly. Once the pH is adjusted to 5.0, the acetyl phosphate is relatively stable and subsequent operations may be carried out more leisurely. Finally, cold water is added to bring the aqueous layer to 500 ml. in volume and the mixture is extracted three times with 500-ml. amounts of ethyl ether to remove the excess isopropenyl acetate. The aqueous portion is carefully adjusted to pH 8.0 with 4.0 N lithium hydroxide, and the turbid solution is centrifuged to remove the insoluble lithium phosphate. The clear supernatant solution is placed in an ice bath, and, with stirring, 5 vol. of cold ethyl alcohol is added slowly. The dilithium monoacetylphosphate which crystallizes out is filtered on a Biichner funnel, washed with absolute ethanol and finally with ether, and then dried in a vacuum desiccator over P205 and paraffin. By this procedure 20 to 45 g. of dilithium a c e t y l ~ P is obtained. Sometimes the product first obtained is only 80 to 90% pure. To obtain an analytically pure product the crude material is further purified by fractional precipitation from a 10% solution by addition of ethanol. The precipitate formed with 0.5 vol. of ethanol contains most of the impurities and is discarded. The pure compound is then precipitated by the slow further addition of 5 vol. of cold ethanol and is collected and dried as described above. Properties. When kept dry and cold (0° to - 10°) the lithium acetyP-~P may be stored for years without appreciable decomposition. Neutral solutions of acetyl~-~P may be stored for months at - 10° without appreciable decomposition. Solutions of the compound are very unstable at low and high pH and at elevated temperatures. Complete hydrolysis occurs in 5 minutes at 100 °.
Preparation of Acetyl~-~P by Reaction of K~HPO4 with Acetic Anhydride (Procedure B) This method was previously described by Avison2~and is based on the fact that acetic anhydride will readily acetylate orthophosphate at neutral to slightly alkaline pH in aqueous pyridine solution. The acetyl I A. W, D. Avison, J. Chem. •oc. 1965, 732.
230
CARBOHYImATES
[$9]
phosphate is isolated as the dilithium salt b y precipitation from dilute alcoholic solution. Reagenls
Pyridine. Dipotassium hydrogen phosphate, 0.25 M. Lithium hydroxide, 4 N. E t h y l alcohol. Procedure. Ninety-five grams of pyridine (1.2 moles)"and 200 ml. of 0.25 M dipotassium hydrogen phosphate solution are stirred together at 0 ° to give a clear homogeneous solution. Then 9.6 ml. of acetic anhydride (0.1 mole) is added dropwise to the vigorously stirred solution over a 5-minute interval during which time the t e m p e r a t u r e m a y rise to 3 ° to 4 °. After 35 minutes 38 ml. of 4 N lithium hydroxide is added cautiously. The final p H (after tenfold dilution with water) should be about 7.6. Cold ethanol (2300 ml.) is then added slowly, and after standing for 2 hours at 0 ° the precipitate is filtered off, washed with absolute ethanol and then with ether, and finally dried in a v a c u u m desiccator over P~05 and paraffin. B y this procedure 6 to 7 g. of dilithium a c e t y l ~ P of about 70% p u r i t y is obtained. A product of higher p u r i t y m a y be obtained b y recrystallization of the dilithium salt as described in procedure A above. Comment. Since the acetylation of orthophosphate b y procedure B is essentially quantitative, it has, with slight modification, been used for the synthesis of P3~-labeled a c e t y l ~ P 2 Assay Method
Principle. This method was previously described b y Lipmann and T u t t l e ~ and is based on the fact t h a t acyl phosphates react rapidly with hydroxylamine at p H 6.5 to 7.0 to form hydroxamic acids, which in the
8 Details of the procedure as modified by Kornberg for the synthesis of Pa2-1abeled acetyl,~P are as follows: pyridine (0.95 ml.), K~HP04 (1 M, 0.5 ml., containing PSS-labeled phosphate as desired), and water (1.5 ml.) are mixed in a 30-ml. Erlenmeyer flask. The mixture is called to 0 ° or less, then kept in ice. Acetic anhydride in slight excess (0.11 ml.) is added over a 3-minute period to the mixture which is constantly shaken. LiOH (4 N) is added 2 minutes later to adjust the pH to about 7.5 (about 4.5 ml. is required). Ethanol (23 ml. at - 1 5 °) is added slowly with agitation of the reaction mixture. After 1 hour at 0 °, the precipitate is collected by centrifugation, washed twice with cold ethanol, and dried in vaeuo over CaCl2 and KOH. About 400 micromoles of acetyl,-~P is recovered, a yield of about 80% based on phosphate. The purity is about 60%. No inorganic orthophosphate is normally detected in the crude product. See A. Kornberg, S. R. Kornberg, and E. S. Simms, Biochim. el Biophys. Acta 20, 215 (1956). 4 F. Lipmann and L. C. Tattle, J. Biol. Chem. 15g, 21 (1945).
[39]
PREPARATION AND ASSAY OF ACETYL PHOSPHATE
231
presence of ferric salts produce red to violet complexes that may be quantitated by colorimetric analysis.
Reagents Neutralized, hydroxylamine solution. Prepare just before use by mixing equal volumes of 28% solution of NH2OH.tIC1 (4.0 M) and 14% NaOH (3.5 M). Ferric chloride solution. Prepare by mixing equal volumes of 5 % FeCl3 (in N/IO HC1), 12% TCA, and 3 N HC1. Hydroxamic acid reference standards: (1) Dissolve 30 mg. of acetohydroxamic acid in 100 ml. of H20 (2.0 ml. of this solution = 4.0 micromoles). (2) Alternatively a hydroxamic acid standard solution may be prepared from succinic anhydride as follows: Dissolve 1.0 g. of succinic anhydride in 40 ml. of the neutralized hydroxylamine solution, and dilute to 100 ml. When 1 ml. of this stock solution is diluted to 40 ml. with water, 2.0 ml. of the resulting solution gives a color equivalent to 4.0 micromoles of acetohydroxamic acid.
Procedure. To 2.0 ml. of test solution containing 0.5 to 5 micromoles of acetyl~-~P, add 1.0 ml. of the neutralized hydroxylamine reagent. After 10 minutes at room temperature, add 3.0 ml. of the ferric chloride reagent. If protein is present, remove it by centrifugation, and read the colored supernatant solution immediately in a colorimeter at 540 m~. The optical density is proportional to the concentration of acetohydroxamic acid over the range of 0 to 6 micromole per 6.0 ml. of final solution. Specificily. Under the mild conditions described the method will measure acid anhydrides, acid chlorides, thiol esters, s acyl phosphates and N-acyl imidazoles. All these compounds give essentially equivalent color values. Ordinary esters, aldehydes, amides, and ketones do not interfere even at relatively high concentrations. Comments. High concentrations of various anions depress color development. Thus, fluoride in concentrations of 100, 50, and 25 micromoles per 6 ml. of final reaction mixture depresses the color development 33, 7, and 0%, respectively. With phosphate, 100 and 50 micromoles depress the color 13 and 4%, respectively, and depression with 200 micromoles of sulfate ion is 8 %. Citrate in comparable concentrations is without effect. Other anions have not been tested. 6Thio esters of ~-keto acids do not react with hydroxylamine to give hydroxamic acids. Instead, pyrazolones are formed which give much less intense colors with the iron reagent.
CARBOHYDRATES
[39]
Bandurski and Axelrod 8 described conditions for the identification of phosphopyruvic acid by two-dimensional paper chromatography. Utter and Kurahashi 9 found that phosphopyruvic acid may be purified efficiently by a passage through a column of Dowex 1 resin. They obtained by this step the acid prepared from the first barium precipitate of the Baer-Fischer synthesis 6 in 50 % yield and in a state of 90% purity. Stability. Silver barium phosphopyruvate is stable for years at room temperature in brown glass containers. A slight discoloration after long storage may be disregarded if the salt is being used for the preparation of phosphopyruvic acid. Drying Conditions. 1 At 78 °, silver barium phosphopyruvate loses, per mole, between 0.9 and 1 mole of its 2 moles of crystal water within 1 hour in high vacuum over P2Os. Drying at 100 ° in a high vacuum results in discoloration and larger loss of weight. s R. S. Bandurski and B. Axelrod, J. Biol. Chem. 193, 405 (1951). 9 M. F. Utter and K. Kurahashi, J. Biol Chem. 207, 821 (1954).
[39] Preparation and Assay of Acetyl Phosphate B y E . R. STADTMAN
Preparation of Acetyl Phosphate (Acetyl~-~P) by Reaction of Orthophosphate with Isopropenyl Acetate (Procedure A) Principle. This method, which has been previously described, ~ is based on the fact that orthophosphoric acid is readily acetylated by isopropenyl acetate to give monoacetyl dihydrogen phosphate. The product is isolated as the dilithium salt by fractional crystallization from dilute alcoholic solution. Reagents 85% sirupy phosphoric acid. Isopropenyl acetate (freshly distilled). Concentrated sulfuric acid. Lithium hydroxide, 4 N. Procedure. Two hundred milliliters of isopropenyl acetate in a 1-1. flask is cooled in an ice bath. Then, with stirring, 25 ml. of 85 % sirupy phosphoric acid is added dropwise, and finally 1.0 ml. of concentrated sulfuric acid is added as catalyst. The reaction mixture is placed in a water bath at 30° and is stirred constantly to prevent localizing heating. l E. R. Stadtman and F. Lipmann, J. Biol. Chem. 185, 549 (1950).
[39]
PREPARATION AND ASSAY OF ACETYL PHOSPHATE
229
After 30 minutes, or when the reaction mixture becomes pale yellow in color, it is cooled in an ice bath and 100 g. of ice is added. The mixture is neutralized to pH 5.0 with cold 4 N lithium hydroxide (about 300 ml.). During neutralization, the mixture may separate into two phases, since the isopropenyl acetate which is present in excess is relatively insoluble in water. It is therefore necessary to shake the mixture vigorously in an ice bath during the addition of lithium hydroxide so as to extract the acetyl phosphoric acid from the organic phase. The neutralization should be carried out as rapidly as possible without, however, allowing the temperature to rise unduly. Once the pH is adjusted to 5.0, the acetyl phosphate is relatively stable and subsequent operations may be carried out more leisurely. Finally, cold water is added to bring the aqueous layer to 500 ml. in volume and the mixture is extracted three times with 500-ml. amounts of ethyl ether to remove the excess isopropenyl acetate. The aqueous portion is carefully adjusted to pH 8.0 with 4.0 N lithium hydroxide, and the turbid solution is centrifuged to remove the insoluble lithium phosphate. The clear supernatant solution is placed in an ice bath, and, with stirring, 5 vol. of cold ethyl alcohol is added slowly. The dilithium monoacetylphosphate which crystallizes out is filtered on a Biichner funnel, washed with absolute ethanol and finally with ether, and then dried in a vacuum desiccator over P205 and paraffin. By this procedure 20 to 45 g. of dilithium a c e t y l ~ P is obtained. Sometimes the product first obtained is only 80 to 90% pure. To obtain an analytically pure product the crude material is further purified by fractional precipitation from a 10% solution by addition of ethanol. The precipitate formed with 0.5 vol. of ethanol contains most of the impurities and is discarded. The pure compound is then precipitated by the slow further addition of 5 vol. of cold ethanol and is collected and dried as described above. Properties. When kept dry and cold (0° to - 10°) the lithium acetyP-~P may be stored for years without appreciable decomposition. Neutral solutions of acetyl~-~P may be stored for months at - 10° without appreciable decomposition. Solutions of the compound are very unstable at low and high pH and at elevated temperatures. Complete hydrolysis occurs in 5 minutes at 100 °.
Preparation of Acetyl~-~P by Reaction of K~HPO4 with Acetic Anhydride (Procedure B) This method was previously described by Avison2~and is based on the fact that acetic anhydride will readily acetylate orthophosphate at neutral to slightly alkaline pH in aqueous pyridine solution. The acetyl I A. W, D. Avison, J. Chem. •oc. 1965, 732.
230
CARBOHYImATES
[$9]
phosphate is isolated as the dilithium salt b y precipitation from dilute alcoholic solution. Reagenls
Pyridine. Dipotassium hydrogen phosphate, 0.25 M. Lithium hydroxide, 4 N. E t h y l alcohol. Procedure. Ninety-five grams of pyridine (1.2 moles)"and 200 ml. of 0.25 M dipotassium hydrogen phosphate solution are stirred together at 0 ° to give a clear homogeneous solution. Then 9.6 ml. of acetic anhydride (0.1 mole) is added dropwise to the vigorously stirred solution over a 5-minute interval during which time the t e m p e r a t u r e m a y rise to 3 ° to 4 °. After 35 minutes 38 ml. of 4 N lithium hydroxide is added cautiously. The final p H (after tenfold dilution with water) should be about 7.6. Cold ethanol (2300 ml.) is then added slowly, and after standing for 2 hours at 0 ° the precipitate is filtered off, washed with absolute ethanol and then with ether, and finally dried in a v a c u u m desiccator over P~05 and paraffin. B y this procedure 6 to 7 g. of dilithium a c e t y l ~ P of about 70% p u r i t y is obtained. A product of higher p u r i t y m a y be obtained b y recrystallization of the dilithium salt as described in procedure A above. Comment. Since the acetylation of orthophosphate b y procedure B is essentially quantitative, it has, with slight modification, been used for the synthesis of P3~-labeled a c e t y l ~ P 2 Assay Method
Principle. This method was previously described b y Lipmann and T u t t l e ~ and is based on the fact t h a t acyl phosphates react rapidly with hydroxylamine at p H 6.5 to 7.0 to form hydroxamic acids, which in the
8 Details of the procedure as modified by Kornberg for the synthesis of Pa2-1abeled acetyl,~P are as follows: pyridine (0.95 ml.), K~HP04 (1 M, 0.5 ml., containing PSS-labeled phosphate as desired), and water (1.5 ml.) are mixed in a 30-ml. Erlenmeyer flask. The mixture is called to 0 ° or less, then kept in ice. Acetic anhydride in slight excess (0.11 ml.) is added over a 3-minute period to the mixture which is constantly shaken. LiOH (4 N) is added 2 minutes later to adjust the pH to about 7.5 (about 4.5 ml. is required). Ethanol (23 ml. at - 1 5 °) is added slowly with agitation of the reaction mixture. After 1 hour at 0 °, the precipitate is collected by centrifugation, washed twice with cold ethanol, and dried in vaeuo over CaCl2 and KOH. About 400 micromoles of acetyl,-~P is recovered, a yield of about 80% based on phosphate. The purity is about 60%. No inorganic orthophosphate is normally detected in the crude product. See A. Kornberg, S. R. Kornberg, and E. S. Simms, Biochim. el Biophys. Acta 20, 215 (1956). 4 F. Lipmann and L. C. Tattle, J. Biol. Chem. 15g, 21 (1945).
[39]
PREPARATION AND ASSAY OF ACETYL PHOSPHATE
231
presence of ferric salts produce red to violet complexes that may be quantitated by colorimetric analysis.
Reagents Neutralized, hydroxylamine solution. Prepare just before use by mixing equal volumes of 28% solution of NH2OH.tIC1 (4.0 M) and 14% NaOH (3.5 M). Ferric chloride solution. Prepare by mixing equal volumes of 5 % FeCl3 (in N/IO HC1), 12% TCA, and 3 N HC1. Hydroxamic acid reference standards: (1) Dissolve 30 mg. of acetohydroxamic acid in 100 ml. of H20 (2.0 ml. of this solution = 4.0 micromoles). (2) Alternatively a hydroxamic acid standard solution may be prepared from succinic anhydride as follows: Dissolve 1.0 g. of succinic anhydride in 40 ml. of the neutralized hydroxylamine solution, and dilute to 100 ml. When 1 ml. of this stock solution is diluted to 40 ml. with water, 2.0 ml. of the resulting solution gives a color equivalent to 4.0 micromoles of acetohydroxamic acid.
Procedure. To 2.0 ml. of test solution containing 0.5 to 5 micromoles of acetyl~-~P, add 1.0 ml. of the neutralized hydroxylamine reagent. After 10 minutes at room temperature, add 3.0 ml. of the ferric chloride reagent. If protein is present, remove it by centrifugation, and read the colored supernatant solution immediately in a colorimeter at 540 m~. The optical density is proportional to the concentration of acetohydroxamic acid over the range of 0 to 6 micromole per 6.0 ml. of final solution. Specificily. Under the mild conditions described the method will measure acid anhydrides, acid chlorides, thiol esters, s acyl phosphates and N-acyl imidazoles. All these compounds give essentially equivalent color values. Ordinary esters, aldehydes, amides, and ketones do not interfere even at relatively high concentrations. Comments. High concentrations of various anions depress color development. Thus, fluoride in concentrations of 100, 50, and 25 micromoles per 6 ml. of final reaction mixture depresses the color development 33, 7, and 0%, respectively. With phosphate, 100 and 50 micromoles depress the color 13 and 4%, respectively, and depression with 200 micromoles of sulfate ion is 8 %. Citrate in comparable concentrations is without effect. Other anions have not been tested. 6Thio esters of ~-keto acids do not react with hydroxylamine to give hydroxamic acids. Instead, pyrazolones are formed which give much less intense colors with the iron reagent.