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[19] d -glucose-6-phosphate
152
CARBOHYDRATES
[19]
for 30 minutes. The silver salts are removed by centrifugation and washed with benzene. After distillation of the solvent in vacuo, the residue of the benzene solutions is dissolved in a small amount of chloroform, reprecipitated with petroleum ether, dried in vacuo, and redissolved in 10 ml. of anhydrous methanol. The filtered solution is hydrogenated in the presence of 200 mg. of platinum oxide. Usually the theoretical amount of hydrogen (16 moles per mole of sugar) is absorbed in 1 hour; if the gas uptake becomes too slow, it is necessary to add 100 mg. more of catalyst. After filtration, normal sodium hydroxide is added to the cooled solution, until a permanent pink color is obtained with phenolphthalein. A gummy precipitate separates which is dissolved by addition of water. The solution is then concentrated in vacuo in order to remove methanol and cyclohexane. The inorganic phosphate which represents about 30% of the total phosphate is precipitated with an excess of magnesia mixture prepared from magnesium chloride. The pH of the filtered solution is adjusted to 8.5 with acetic acid. By addition of 0.5 g. of barium acetate and heating to boiling for 2 minutes, the barium salt is precipitated; it is centrifuged while hot and washed with hot water, alcohol, and ether. The compound (115 rag.), which already consists mainly of a-glucose-l,6diphosphate, is purified as brucine salt as indicated above. Sodium salt: [a]~s = +80.3 ° +_ 2.4 ° (water, c = 0.406, calculated as anhydrous free acid in water). When ethanol instead of methanol is the solvent for the hydrogenation, the amount of inorganic phosphate formed is about twice as small and the final yield is twice as great. However the isolated glucose-l,6diphosphate contains about 25 % of the f~ form, and the separation of the components has to be carried out by fractional crystallization of the brucine salts as described above.
[19] D-Glucose-6-phosphate By B. L. HORECKER and W. A. WOOD
Preparation Principle. Fructose-6-phosphate is converted to glucose-6-phosphate by the action of hexose phosphate isomerase. In the presence of barium ion the equilibrium
D-Fructose-6-phosphate ~-~ D-Glucose-6-phosphate is displaced by the precipitation of a highly insoluble barium glucose6-phosphate heptahydrate, and the reaction proceeds essentially to completion.
[19]
D-GLUCOSE-6-PHOSPHATE
153
Reagents. Fructose-6,phosphate may be obtained by acid hydrolysis 1 of fructose-l,6-diphosphate. ~ Commercial barium fructose-6-phosphate, which contains about 40% of barium fructose-6-phosphate and 15% of barium glucose-6-phosphate, is an inexpensive starting material. Active preparations of hexose phosphate isomerase may be prepared from rat or rabbit skeletal muscle or from bacterial extracts. The enzyme preparations must be freed from sulfate and phosphate ions by dialysis. A crude hexose phosphate isomerase from muscle is adequate for the preparation (for the preparation of the purified enzyme, see Vol. I [37]). Fresh rat skeletal muscle is ground with a small hand meat grinder, extracted with an equal weight of cold water, and strained through several layers of cheesecloth. The residue is again extracted with one-half the original quantity of water. The combined extracts are dialyzed against cold distilled water until free of inorganic phosphate2 The dialyzed solution is centrifuged for 10 minutes at 18,000 × g, and the precipitate is discarded. Procedure. Two grams of commercial barium fructose-6-phosphate is dissolved in 20 ml. of water, and the insoluble residue is removed by centrifugation. 4 The supernatant solution, containing approximately 0.68 millimole of glucose-6-phosphate and 1.74 millimoles of fructose-6phosphate, is treated with 4.0 ml. of the dialyzed isomerase preparation. Several seed crystals are added, and the reaction is allowed to proceed at room temperature. After 16 hours the mixture is.chilled to 0 ° and filtered. The crystalline precipitate is washed with a small volume of cold water and dried in air. The product weighs 1.0 g. and contains 1.92 millimoles of barium glucose-6-phosphate heptahydrate (79% of theory). It may be dissolved in hydrochloric acid and recrystallized by neutralizing the solution. Properties
The properties of glucose-6-phosphate and several methods of analysis have been described previously. 5 Assay. A sensitive and highly specific method for the determination of glucose-6-phosphate is the reduction of triphosphopyridine nucleotide 1C. Neuberg, H. Lustig, and M. A. Rothenberg, Arch. Biochem. 3, 33 (1943); see Vol. III [22]. 2 H. Z. Sable, Biochem. Preparations % 52 (1952); see Vol. III [22]. 8 An 8-hour dialysis period is satisfactory. 4Some commercial batches of fructose-6-phosphate give a deeply colored yelloworange solution and, in this procedure, yield appreciable quantities of an amorphous product which retains the chromogen. Such batches may be treated with activated carbon and filtered while hot to give clear solutions which yield only the crystalline product. s H. A. Lardy, Biochem. Preparations 2, 39 (1952).
CARBOHYDRATES
154
[20]
( T P N ) in the presence of glucose-6-phosphate dehydrogenase. B T h e a m o u n t of glucose-6-phosphate m a y be calculated from the change in optical density at 340 m~. 7 Into a 1.0-cm. absorption cell are pipetted 0.65 ml. of water, 0.15 ml. of 0.25 M glycylglycine buffer, p H 7.5, 0.10 ml. of 0.1 M MgC12, 0.10 ml. of T P N (1.4 ~M./ml.), and 0.02 ml. of glucose6-phosphate dehydrogenase s (ca. 0.02 rag.). The solution is mixed, and an initial density reading at 340 m~ is taken. The glucose-6-phosphate sample to be analyzed, containing 0.02 to 0.10 micromole in 0.02 ml., is added, and the increase in density at 340 m~ is followed until a constant value is reached. The a m o u n t of glucose-6-phosphate added is calculated from the relation G-6-P (micromoles) = ( d / -
do) X 1.04 6.22
where d / a n d do are the final and initial density readings, respectively. Analysis. The p u r i t y of the crystalline barium salt is 98 to 102% b y this enzymatic assay procedure without recrystallization. The analytical composition is as tabulated. For C6HllOgPBa.7H20 Ba P H20 ' Calculated, % 26.2 5.94 24.1 Found, % 25.9 5.79 24.4 Unlike the amorphous product, the crystalline h e p t a h y d r a t e is highly insoluble in water. The ~olubility is approximately 2 X l0 -8 M. at 25 ° and 1 X 10-3 M. at 0 °. I t is readily soluble in dilute acid. s A. Kornberg, J. Biol. Chem. 182, 805 (1950); see Vol. I [42]. 7 B. L. Horecker and A. Kornberg, J. Biol. Chem. 175, 385 (1948). 8 See Vol. I [42]. ' Weight loss at 70° in high vacuum.
[20] S y n t h e s i s
of M a n n o s e - 6 - p h o s p h a t e
by Hexokinase
B y MILTON W. SLEIN Preparation
The use of crystalline yeast hexokinase 1 for catalyzing the phosphorylation of mannose b y A T P will result in a highly pure preparation of mannose-6-phosphate (M-6-P). A product contaminated with only traces 1 For the preparation of hexokinase, see Vol. I [32]. Noncrystalline, partially purified preparations are commercially available. It would be necessary to test these for phosphomannose isomerase (PMI) activity before use (see Vol. I [37]). If present, the PMI should be inactivated as described below for crude yeast extract.
CARBOHYDRATES
[19]
for 30 minutes. The silver salts are removed by centrifugation and washed with benzene. After distillation of the solvent in vacuo, the residue of the benzene solutions is dissolved in a small amount of chloroform, reprecipitated with petroleum ether, dried in vacuo, and redissolved in 10 ml. of anhydrous methanol. The filtered solution is hydrogenated in the presence of 200 mg. of platinum oxide. Usually the theoretical amount of hydrogen (16 moles per mole of sugar) is absorbed in 1 hour; if the gas uptake becomes too slow, it is necessary to add 100 mg. more of catalyst. After filtration, normal sodium hydroxide is added to the cooled solution, until a permanent pink color is obtained with phenolphthalein. A gummy precipitate separates which is dissolved by addition of water. The solution is then concentrated in vacuo in order to remove methanol and cyclohexane. The inorganic phosphate which represents about 30% of the total phosphate is precipitated with an excess of magnesia mixture prepared from magnesium chloride. The pH of the filtered solution is adjusted to 8.5 with acetic acid. By addition of 0.5 g. of barium acetate and heating to boiling for 2 minutes, the barium salt is precipitated; it is centrifuged while hot and washed with hot water, alcohol, and ether. The compound (115 rag.), which already consists mainly of a-glucose-l,6diphosphate, is purified as brucine salt as indicated above. Sodium salt: [a]~s = +80.3 ° +_ 2.4 ° (water, c = 0.406, calculated as anhydrous free acid in water). When ethanol instead of methanol is the solvent for the hydrogenation, the amount of inorganic phosphate formed is about twice as small and the final yield is twice as great. However the isolated glucose-l,6diphosphate contains about 25 % of the f~ form, and the separation of the components has to be carried out by fractional crystallization of the brucine salts as described above.
[19] D-Glucose-6-phosphate By B. L. HORECKER and W. A. WOOD
Preparation Principle. Fructose-6-phosphate is converted to glucose-6-phosphate by the action of hexose phosphate isomerase. In the presence of barium ion the equilibrium
D-Fructose-6-phosphate ~-~ D-Glucose-6-phosphate is displaced by the precipitation of a highly insoluble barium glucose6-phosphate heptahydrate, and the reaction proceeds essentially to completion.
[19]
D-GLUCOSE-6-PHOSPHATE
153
Reagents. Fructose-6,phosphate may be obtained by acid hydrolysis 1 of fructose-l,6-diphosphate. ~ Commercial barium fructose-6-phosphate, which contains about 40% of barium fructose-6-phosphate and 15% of barium glucose-6-phosphate, is an inexpensive starting material. Active preparations of hexose phosphate isomerase may be prepared from rat or rabbit skeletal muscle or from bacterial extracts. The enzyme preparations must be freed from sulfate and phosphate ions by dialysis. A crude hexose phosphate isomerase from muscle is adequate for the preparation (for the preparation of the purified enzyme, see Vol. I [37]). Fresh rat skeletal muscle is ground with a small hand meat grinder, extracted with an equal weight of cold water, and strained through several layers of cheesecloth. The residue is again extracted with one-half the original quantity of water. The combined extracts are dialyzed against cold distilled water until free of inorganic phosphate2 The dialyzed solution is centrifuged for 10 minutes at 18,000 × g, and the precipitate is discarded. Procedure. Two grams of commercial barium fructose-6-phosphate is dissolved in 20 ml. of water, and the insoluble residue is removed by centrifugation. 4 The supernatant solution, containing approximately 0.68 millimole of glucose-6-phosphate and 1.74 millimoles of fructose-6phosphate, is treated with 4.0 ml. of the dialyzed isomerase preparation. Several seed crystals are added, and the reaction is allowed to proceed at room temperature. After 16 hours the mixture is.chilled to 0 ° and filtered. The crystalline precipitate is washed with a small volume of cold water and dried in air. The product weighs 1.0 g. and contains 1.92 millimoles of barium glucose-6-phosphate heptahydrate (79% of theory). It may be dissolved in hydrochloric acid and recrystallized by neutralizing the solution. Properties
The properties of glucose-6-phosphate and several methods of analysis have been described previously. 5 Assay. A sensitive and highly specific method for the determination of glucose-6-phosphate is the reduction of triphosphopyridine nucleotide 1C. Neuberg, H. Lustig, and M. A. Rothenberg, Arch. Biochem. 3, 33 (1943); see Vol. III [22]. 2 H. Z. Sable, Biochem. Preparations % 52 (1952); see Vol. III [22]. 8 An 8-hour dialysis period is satisfactory. 4Some commercial batches of fructose-6-phosphate give a deeply colored yelloworange solution and, in this procedure, yield appreciable quantities of an amorphous product which retains the chromogen. Such batches may be treated with activated carbon and filtered while hot to give clear solutions which yield only the crystalline product. s H. A. Lardy, Biochem. Preparations 2, 39 (1952).
CARBOHYDRATES
154
[20]
( T P N ) in the presence of glucose-6-phosphate dehydrogenase. B T h e a m o u n t of glucose-6-phosphate m a y be calculated from the change in optical density at 340 m~. 7 Into a 1.0-cm. absorption cell are pipetted 0.65 ml. of water, 0.15 ml. of 0.25 M glycylglycine buffer, p H 7.5, 0.10 ml. of 0.1 M MgC12, 0.10 ml. of T P N (1.4 ~M./ml.), and 0.02 ml. of glucose6-phosphate dehydrogenase s (ca. 0.02 rag.). The solution is mixed, and an initial density reading at 340 m~ is taken. The glucose-6-phosphate sample to be analyzed, containing 0.02 to 0.10 micromole in 0.02 ml., is added, and the increase in density at 340 m~ is followed until a constant value is reached. The a m o u n t of glucose-6-phosphate added is calculated from the relation G-6-P (micromoles) = ( d / -
do) X 1.04 6.22
where d / a n d do are the final and initial density readings, respectively. Analysis. The p u r i t y of the crystalline barium salt is 98 to 102% b y this enzymatic assay procedure without recrystallization. The analytical composition is as tabulated. For C6HllOgPBa.7H20 Ba P H20 ' Calculated, % 26.2 5.94 24.1 Found, % 25.9 5.79 24.4 Unlike the amorphous product, the crystalline h e p t a h y d r a t e is highly insoluble in water. The ~olubility is approximately 2 X l0 -8 M. at 25 ° and 1 X 10-3 M. at 0 °. I t is readily soluble in dilute acid. s A. Kornberg, J. Biol. Chem. 182, 805 (1950); see Vol. I [42]. 7 B. L. Horecker and A. Kornberg, J. Biol. Chem. 175, 385 (1948). 8 See Vol. I [42]. ' Weight loss at 70° in high vacuum.
[20] S y n t h e s i s
of M a n n o s e - 6 - p h o s p h a t e
by Hexokinase
B y MILTON W. SLEIN Preparation
The use of crystalline yeast hexokinase 1 for catalyzing the phosphorylation of mannose b y A T P will result in a highly pure preparation of mannose-6-phosphate (M-6-P). A product contaminated with only traces 1 For the preparation of hexokinase, see Vol. I [32]. Noncrystalline, partially purified preparations are commercially available. It would be necessary to test these for phosphomannose isomerase (PMI) activity before use (see Vol. I [37]). If present, the PMI should be inactivated as described below for crude yeast extract.