[33] L-0~-GLYCEROPHOSPHATE 207

pounds and repeated standardization. In addition, the method can'0nly yield linear activity vs. enzyme concentration curves over a limited range of enzyme concentrations, owing to the limitation set by the magnitude of the ratio between the eM values of the DHA-P and G-3-P chromogens.

Separation of Triose Phosphates

In tissue fractionation, triose phosphates appear in the barium- soluble-alcohol-precipitable fraction. In preparing substrates for enzyme experiments, some of the earlier workers used this method and another method involving hydrazine interception in large-scale aldolase incuba- tions, followed by benzaldehyde and ether extraction. More recently, a variety of chromatographic procedures have appeared in the litera- ture, references to which may be found in reviews by Block et al. ~5 and Isherwood. ~6 2s 1~. Block, E. Durrum, and G. Zweig, "A Manual of Paper Chromatography and

Paper Electrophoresis," 2nd ed. Academic Press, New York~ 1955. ~e F. A. Isherwood, Brit. Med. Bull. 10, 202 (1954).

[33] L - a - G l y c e r o p h o s p h a t e

By HENRY LARDY

H2COH

l HOCH

I H~COPO3Ba

The synthesis of the natural isomer of a-glycerophosphate is accom- plished by the procedure of Baer and Fischer. I The reaction is carried out in a two-necked flask fitted with a dropping funnel and a vent through a calcium chloride tube. The flask is cooled at all times by a salt-ice bath at -- 15 °.

A solution of 8.75 g. of D-isopropylidene-glycerol ~ in 24 ml. of pure, dry quinoline is added dropwise with shaking during a period of 20 min- utes to a solution of 10.5 g. of phosphorus oxychloride in 30 ml. of quino- line. 3 After 1 hour the flask may be removed from the cold bath and 1 E. Baer and H. O. L. Fischer, J. Biol. Chem. 128, 491 (1939). E. Baer and H. O. L. Fischer, J. Biol. Chem. 128, 463 (1939); E. Baer, Biochem. Preparations 2, 31 (1952).

3 This solution is prepared by adding phosphorus oxychloride dropwise to precooled quinoline.

208 CARBOHYDRATES [34]

permitted to come to room temperature to complete the reaction and to facilitate handling the mixture which is extremely viscous at the low temperature of the bath. The reaction mixture is then poured slowly into a vigorously stirred mixture of 30 ml. of H2SO4, 28 g. of Ag2CO3, and 700 ml. of ice and water. After 30 minutes of stirring the solution is filtered through a layer of charcoal on a precoated funnel and silver ion is removed from the solution by treatment with H2S. The clear filtrate is heated in a boiling water bath for 30 minutes to free it of H2S and to remove the isopropylidene group. The solution is cooled to 0 ° and, with stirring, finely pulverized Ba(OH)~'8H20 (about 100 g. is required) is added slowly until the solution becomes alkaline to phenolphthalein. Excess barium ion is removed with carbon dioxide and, after filtration, the aqueous solution is concentrated by distillation in the vacuum of a water pump and with a bath at 40 °. When the volume of the solution has been reduced to about 60 ml., a slight turbidity is removed by filtration. The barium salt of ~-glycerophosphate is precipitated from solution by heating in a boiling water bath for about 20 minutes and collected, while hot, on a hard paper or sintered glass Biichner funnel. After drying i n vacuo over P~Os, the compound has the composition of the anhydrous barium salt (molecular weight 307.5). Yields of 30 to 37 % of theoretical are obtained.

For biological work the barium salt is dissolved in water with the aid of a little acid. Sodium or potassium sulfate is then added, and after removal of the precipitated BaSO4 the solution is brought to the desired pH and volume.

The compound is extremely resistant to hydrolysis by the standard analytical procedure (1 N HC1, 100°). In hot alkaline solution the phos- phate group migrates to the ~ position and optical activity is lost.

[34] Preparation of D(- )-3-Phosphoglyceric Acid

By INES MANDL a n d CARL NEUBERG

Natural 3-PGA may be prepared by three different methods: 1. I t may be prepared biologically from yeast and sugar phosphate

solution in the presence of acetaldehyde or another hydrogen acceptor and fluoride. ~-4 Such substances prevent a simultaneous formation of I C. Neuberg and M. Kobel, Biochem. Z. 268, 219 (1933). 2 C. Neuberg and M. Kobel, Biochem. Z. 264, 456 (1933). I C. Neuberg and M. Kobel, Angew. Chem. 46, 711 (1933). 4 C. Neuberg and H. Lustig, Arch. Biochem. 1, 311 (1942).