STUDIES OF PHOSPHORUS OF BLOOD

V. A COMPARATIVE STUDY OF ACID AND ENZYMATIC HYDROLY- SIS OF THE ACID-SOLUBLE ORGANIC PHOSPHORUS,

WITH PARTICULAR REFERENCE TO THE PHOSPHOGLYCERATE FRACTION

BY EDNA WARWEG AND GENEVIEVE STEARNS

(From the Department oj Pediatrics, College of Medicine, State University of Iowa, Iowa City)

(Received for publication, June 8, 1936)

About 60 per cent of the phosphorus of blood consists of organic compounds soluble in dilute acid, the so called acid-soluble organic or ester phosphorus. This fraction of blood phosphorus has been further separated into at least three components, two of which, a pyrophosphate (l-8) and a phosphoglycerate (9, lo), have been isolated from blood. The remainder is thought to be composed of one or more hexosephosphates (1, 11-13).

The above compounds have been studied also by means of their relative rates of hydrolysis, either with acid or with enzymes. It has been shown that the pyrophosphate is completely hy- drolyzed by boiling acid within 7 to 15 minutes (2, 14-17), that the hexosephosphate fraction is less readily hydrolyzed (16, 17), and that the phosphoglycerate is very resistant to hydrolysis (9, 10). Unfortunately, conditions chosen by the various investi- gators have varied so widely that considerable uncertainty still exists, particularly in regard to the relative rates of hydrolysis of the hexosephosphate and phosphoglycerate fractions. Investiga- tions of the enzyme hydrolysis agree only in that all the phosphoric esters can be split to some extent by phosphatase (10, 18-23). It seemed advisable, therefore, to study the complete hydrolysis of the ester phosphorus fraction of blood under controlled and readily reproducible condit,ions. The hydrolysis of phospho- glycerabe isolated from pig blood and of the commercial products fi-glycerophosphate and hexosediphosphate (candiolin) was studied

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568 Blood Phosphorus. V

under the same conditions as was blood. The commercial prep- arations mentioned serve as readily available controls.

EXPERIMENTAL

Normal adults were used to furnish the blood filtrate for the investigation. Into 80 cc. of ice-cold 4 per cent trichloroacetic acid in a 100 cc. volumetric flask, blood was allowed to run directly from the vein to the volume mark. Thus no anticoagulant was necessary and this concentration of trichloroacetic acid, while adequate for precipitation of the protein at the temperature used, was sufficiently low to avoid the inhibitory effect of the trichloro- acetate ions on enzyme hydrolysis (23). Phosphoglyceric acid, as the barium salt, was obtained from pig blood according to the method of Greenwald (9) and converted into the sodium salt which is readily soluble in water. The hexosediphosphatel and P-glycerophosphate were commercial preparations.

The solutions of phosphoglycerate, hexosediphosphate, and Bglycerophosphate were made to simulate the blood filtrate as closely as possible. Because sodium sulfate was a contaminant of the phosphoglycerate and affects the rate of enzyme hydrolysis, it was added in equal concentration to the other solutions. 100 cc. of each solution contained 4 to 8 mg. of organic phosphorus, 0.5 to 1.0 mg. of inorganic phosphorus added as sodium hydrogen phosphate, 4 mg. of sodium sulfate, and 3.2 per cent of trichloro- acetic acid. Aliquots of these solutions, the blood filtrate, phosphoglycerate, /?-glycerophosphate, and hexosediphosphate were used in all subsequent experiments, both acid and enzyme hydrolysis.

For acid hydrolysis, hard glass test-tubes, 250 X 20 mm., graduated at 10 and 20 cc., were fitted with small glass con- densers. The condensers, ground to fit the tubes, served as stoppers, preventing evaporation and eliminating the possibility of increased pressure which occurs when tubes are sealed and immersed in boiling water as advocated by some investigators. 2 cc. aliquots of the solutions under investigation were pipetted into test-tubes, 0.5 cc. of 10 N sulfuric acid added to each, and the walls of each tube washed down with exactly 2.5 cc. of distilled

1 Provided through the courtesy of the Winthrop Chemical Com- pany, Inc.

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E. Warweg and G. Stearns

water. The condensers were fitted and the tubes were placed in a water bath heated to 100’ by means of an electric plate. The bath and electric plate were large enough to keep the temperature constant. At definite time intervals, tubes were removed, cooled in iced water, and the phosphorus determined (24). The total phosphorus in the solution, the inorganic phosphorus at zero time and at the chosen time interval were determined throughout all the experiments according to the method given in a previous publica- tion (25). From the total organic phosphorus and the inorganic phosphorus formed from it during the course of acid hydrolysis, the per cent of hydrolysis at a given time was determined.

For enzyme hydrolysis, phosphatase was prepared from lamb kidney according to the method recommended by Kay (26), the enzyme being extracted from kidney by grinding with sand and water saturated with chloroform. The activity of the phos- phatase thus prepared was standardized (26), 1 unit being the amount of enzyme which at 38” in Sijrensen’s buffer at pH 8.9 liberates 1 mg. of phosphorus from excess /3-glycerophosphate in 2 hours. The extract of phosphatase was diluted to contain 150 units per 100 cc. and the same concentration of enzyme was used throughout all the experiments. The activity was checked during each experiment since phosphatase is not stable.

Enzymatic hydrolysis was carried out, at pH 8.8, which has been reported optimal for kidney phosphatase (19, 26, 27), and also at pH 7.4 to approximate the pH of the blood. The pH of the substrate to be used, blood filtrate, phosphoglycerate, P-gly- cerophosphate, or hexosediphosphate was adjusted first with solid NaOH, then an equal volume of sodium diethylbarbiturate buffer solution (28) of the desired pH was added, and the resulting solu- tion well mixed. In each of the series of tubes were placed 10 cc. of the substrate buffer solution, 1 cc. of phosphatase extract, and 1 drop of chloroform. Two tubes of solution were used as con- trols, and the remainder were stoppered and placed in an incuba- tor, kept at exactly 38”. The contents of the control tubes were treated immediately with 4 cc. of 10 per cent trichloroacetic acid and filtered. Total phosphorus and inorganic phosphorus were determined in duplicate on samples from each tube, cor- rection being made for the phosphorus present as an impurity in the phosphatase extract. At each desired interval of time, two

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tubes were removed from the incubator, the phosphatase in the solution precipitated by the addition of trichloroacetic acid, and the inorganic and total phosphorus determined on aliquots of the filtrate. From the total organic phosphorus and the inorganic phosphorus formed by the action of the enzyme on the organic phosphorus, the per cent of hydrolysis was determined. Experi- ments were conducted in this way for all solutions at both pH 8.8 and pH 7.4.

In these investigations with phosphatase, effort was made to carry out the experiments under comparable conditions rather than under optimum conditions; hence, from the data one cannot state the hydrogen ion or salt concentration most favorable for the phosphatase activity on a particular substrate. The concen- tration of trichloroacetate ion during the time the enzyme was active was 1.4 per cent, a concentration below that found to inhibit the activity of the enzyme (23). Magnesium has been reported to alter the activity of phosphatase (27, 29, 30) but the magnesium content of the solutions studied was not determined, inasmuch as it would probably be approximately the same in all of the solutions. These solutions contained a relatively high con- centration of sodium sulfate. This salt has a retarding effect on phosphatase, as was determined by comparing the relative rates of hydrolysis of solutions containing varying amounts of sodium sulfate.

In agreement with most investigators, we did not find phospho- creatine in blood. No difference was observed in the inorganic phosphate content of blood whether the blood was allowed to flow into iced trichloroacetic acid and the determination carried out immediately, or whether trichloroacetic acid was added at room temperature and the resulting mixture allowed to stand for 15 to 20 minutes before filtering. In a trichloroacetic acid solution, pyrophosphate apparently does not disintegrate into the inorganic ortho form during this time. The total phosphorus and inorganic phosphorus values of both the warm and cold solutions were the same; therefore phosphocreatine was considered absent from the blood.

The results of the hydrolysis of blood filtrate, phosphoglyccrate, hexosediphosphate, and /3-glycerophosphate with N sulfuric acid at 100” are shown in Chart I. Boiling blood filtrate with acid

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E. Warweg and G. Stearns 571

induced a very rapid breakdown of the organic phosphorus com- pounds during the first 10 minutes. The 11.4 per cent split within this time probably represents, principally, the cleavage of pyrophosphate. After this initial rapid hydrolysis, the rate de- creased progressively until the 12th hour. Then hydrolysis proceeded at a slow but constant velocity, indicating that all the organic phosphorus compounds in the blood filtrate are destroyed

TIME IN HOURS

CHART I. The hydrolysis of the phosphoric esters of blood filtrate, of blood phosphoglycerate, of p-glycerophosphate, and of hexosediphosphate with N HzSOa at 100”.

by the 12th hour with the exception of one which is fairly resistant to acid. According to the literature, this compound is said to be a phosphoglycerate.

The rate of hydrolysis of the phosphoglycerate isolated from pig blood was identical with that of the blood filtrate after the latter had been hydrolyzed for 12 hours. After this time, the organic phosphorus remaining in the blood filtrate and the phos- phoglycerate undissociated after 12 hours of acid treatment are

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572 Blood Phosphorus. V

broken down in the same manner by further hydrolysis. This is shown in Chart II. The similarity of the two compounds can be most clearly demonstrated by analyzing, from the standpoint of chemical kinetics, the hydrolysis rates of the phosphoglycerate isolated from pig blood and the organic phosphorus compound remaining in the filtrate from human blood after 12 hours hy- drolysis. The time required to complete a certain fraction of a reaction is dependent upon the order of that reaction. If the

. /

9

0 BLOOD FILTRATE + PHOSPHOGLYCERATE

9

1, I I I 12 36 60 a4

TIME IN HOURS

CHART II. The hydrolysis of the organic phosphorus of blood filtrate and of the blood phosphoglycerate with N HzS04 at 100” after 12 hours pre- liminary acid hydrolysis.

order of the reaction is monomolecular, the value of K, the velocity constant, is independent of the initial concentration and the formula dx/dt = K(A -s) can be used. By this formula, the average K for the phosphoglycerate isolated from pig blood is 0.0251. The formula does not hold for blood filtrate until after the 12th hour (Table I). From then on, K is 0.0253, showing that the compound left in the filtrate from human blood after 12 hours hydrolysis is identical with the phosphoglycerate actually isolated from pig blood in that both compounds apparently

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E. Warweg and G. Stearns 573

hydrolyze in a monomolecular manner. The order of these reac- tions may be shown graphically also. Plotting the log of the concentration (A - x) against time should give a straight line if a reaction is of the first order. A straight line is obtained by plotting the data from the acid hydrolysis of the phosphoglycerate isolated from blood. The data for blood filtrate after the 12th

TABLE I

Velocity Constants of Acid Hydrolysis of Phosphoric Esters of l3loodPiltrate and of Blood Phosphoglycerate As Determined by the Formula,

dx/dt = K(A - x)

Material hydrolyzed TiUle

Blood filtrate

Phosphoglycerate

hrs.

0

0.17 2 5

12 24 48 72 96

0 0.5 2 5

12 24 48 72 96

2, per cent hydrolyzed

(A - 2) (loo - per

:ent hydro- lyzed)

, _

0 100 11.4 88.6 24.8 75.2 33.9 66.1 48.1 51.9 62.3 37.7 79.7 20.3 89.0 11.0 94.1 5.9

0 100 1.2 98.8 4.8 95.2

11.9 88.1 25.6 74.4 45.1 54.9 70.4 29.6 84.2 15.8 91.6 8.4

xc I Jog (A - 2)

0.7200 0.0882 0.0428 0.0344 0.0264 0.0250 0.0247 0.0251

0.0242 0.0255 0.0258 0.0241 0.0252 0.0249 0.0254 0.0254

2.000 1.947 1..876 1.820 1.715 1.576 1.308 1.041 0.771 2.000 1.995 1.979 I .945 1.872 1.740 I.471 1.199 0.924

hour of hydrolysis not only give a straight line but the line parallels that given by phosphoglycerate (Chart III).

The above results demonstrate that both the isolated compound and the acid-resistant compound of human blood seem to be identical and that their hydrolyses proceed in the same manner as would the hydrolysis of a monophospho ester. Greenwald (Q) concluded from chemical analysis that the phosphoglycerate isolated from pig blood contained 23 per cent of phosphorus and

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574 Blood Phosphorus. V

was a diphospho ester. Chemical analysis of the substance used in the studies reported herein was unsatisfactory because of the difficulty in drying it. If the phosphoglycerate is a diphospho ester, as the chemical evidence of Greenwald indicates, the mono- molecular character of the hydrolysis suggests that the two phosphoric acid groups are split off at approximately the same rate.

1.6 -

52 PHOSPHOCLYCERATE

k

:: -I 1.2 -

I I I BLOOD FILTRATE I

0.8 - I I I I I I I

0 12 24 48 72 96

TIME IN HOURS

CHART III. The order of the reaction of the acid hydrolysis of the phosphoric esters of blood filtrate and of blood phosphoglycerate as det,er- mined by plotting the log of the concentration (A - 2) against time.

The amount of phosphoglycerate normally present in human blood may be determined from the hydrolysis rates of the blood filtrate and of the phosphoglycerate from the blood. The hy- drolysis rates of the two solutions have shown that a single organic phosphorus compound is left in blood filtrate after 12 hours acid hydrolysis, that this compound is split at a constant rate equal to the rate of cleavage of phosphoglycerate, and that the compound will react to acid from the beginning of hydrolysis in a way iden- tical with the phosphoglycerate. Thus, from the amount of

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E. Warweg and G. Stearns 575

phosphorus left in the blood filtrate at any interval of time during the course of hydrolysis, after the 12th hour, and the amount of phosphoglycerate remaining unchanged at the same interval of time, the per cent of blood phosphorus difficultly split with acid can be determined. For example, if the ratio of undissociated organic phosphorus at 48 hours to the undissociated organic phos- phorus at zero time is the same in each solution, then the ratio for pure phosphoglycerate is 29.6: 100 (Table I) and the ratio for blood filtrate must be 20.3 :X or 20.3:68.5. Bloods from forty- five normal human adults were studied and the per cent of hy- drolysis determined at various intervals of time after 12 hours. An average value of 68 per cent phosphoglycerate phosphorus was observed, with a range of 62 to 75 per cent.

Jost (10) has reported 75 to 80 per cent of phosphoglycerate in human blood, Kay and Robison (18) 64 to 86 per cent, Bomskov (17) 56 per cent, and Greenwald (9) 22 per cent. The method of obtaining these values has been different in each case. Green- wald isolated the material as the barium salt and Jost isolated the brucine salt. Incomplete isolation or incomplete separation of the various insoluble organic phosphorus compounds would intro- duce error. Bomskov, using acid hydrolysis, assumed that all the organic phosphorus remaining after 3 hours was probably a phosphoglycerate and that none of it was attacked during the 3 hour period. Kay and Robison considered the fraction resist- ant to enzyme hydrolysis at pH 8.8 to be the phosphoglycerate, but Roche (19) has shown that no phosphorus compound in the blood is absolutely impervious to some action by phosphatase.

Assuming that about 68 per cent of the organic phosphorus in blood filtrate is in the form of phosphoglycerate and that the pyrophosphate is destroyed within 10 minutes, blood from human adults containing an average of 21.2 mg. of ester phosphorus per 100 cc. (25) could be fractionated into 68 per cent or 14.4 mg. of phosphoglycerate phosphorus and 11.4 per cent or 2.4 mg. of pyrophosphate phosphorus (Table I). The remainder, 10.6 per cent or 4.4 mg., would be the fraction at present designated as hexosephosphate phosphorus. Mai (13) and Lawaczek (31) found about 1 per cent hexosephosphate in blood. Such a method of fractionating the organic acid-soluble phosphorus compound of blood is subject to some error and the method has been tried only

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576 Blood Phosphorus. V

with blood of normal adults, but it may aid in determining gross changes in the ester fraction of blood in diseases wherein the phosphorus metabolism is altered.

The rate of hydrolysis of the @-glycerophosphate was much slower than that of the blood phosphoglycerate; the commercial hexosediphosphate was completely hydrolyzed within 9 hours, the “hexosephosphate” fraction of the blood, within 12 hours.

Enzyme Hydrolysis

The hydrolysis rates of the compounds possibly present in blood filtrate were determined also by means of phosphatase. (Charts IV and V). The phosphoglycerate isolated from blood was hydrolyzed with much greater facility at pH 7.4 than at pH 8.8. Asakawa (32) has reported that the optimum pH for the hydrolysis of the phosphoglycerate in blood is 7.7. The organic phosphorus compounds of the blood filtrate also were hydrolyzed more rapidly at pH 7.4 but the amount of inorganic phosphorus liberated in the blood samples as a group varied widely, especially after 24 hours. Despite the range of values, it is significant that the esters of each individual blood were hydrolyzed more rapidly at pH 8.8 during the first 24 hours, then more rapidly at 7.4. This suggests that part of the organic phosphorus compounds of blood filtrate are more easily broken down at pH 8.8 and that after these particular compounds are almost wholly destroyed, a compound is left which, like phos- phoglycerate, is more readily hydrolyzed at pH 7.4. The results of enzyme hydrolysis thus support the acid hydrolysis findings. Roche (19) observed that phosphatase from blood will hydrolyze blood esters and phosphoglyceric acid at the same optimum pH. The results obtained in the present investigation are compatible with those of Kay (26) who conducted experiments for 3 hours and found that the blood filtrate esters were split by phosphatase more easily at pH 8.8. The results are also compatible with the conclusion of King (20) t’hat two groups of esters exist in blood, one of which is more easily hydrolyzed near neutrality and the other, in an alkaline medium.

The experiments with phosphatase hydrolysis of fi-glyccro- phosphate and hexosediphosphate confirm the findings of others (13, 26, 27, 33). Both commercial compounds are split so much

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E. Warweg and G. Stearns 577

more rapidly at either pH than are the phosphorus compounds in the blood filtrate at any time during the course of enzyme hydrolysis that the existence of large amounts of these readily hydrolyzable substances in blood is improbable. While investi- gat.ions with enzymes have contributed to an understanding of the nature of the phosphoric esters in blood, no adequate method has been established whereby these esters can be separated.

,oo _ HEXOSEDIPHOSPHATE

&l-GLYCEROPHOSPHATE

F’HOSPHOGLYCERATE

O- I I I I I

02 5 I2 24 48 72 96

TIME IN HOURS

CHART IV. The hydrolysis of the phosphoric esters of blood filtrate, of blood phosphoglycerate, of fl-glycerophosphate, and of hcxoscdiphos- phate with kidney phosphatase at 38” and at pH 8.8.

From the previous discussion of acid hydrolysis, it has been demonstrated that prolonged heating with acid destroys all but one organic phosphorus compound in the blood filtrate and that this residual compound may be identical with the phosphoglycerate isolated from pig blood. Blood filtrate hydrolyzed with acid for 24 hours might be expected to respond to enzyme treatment as does the isolated phosphoglycerate.

To test the validity of such a premise, blood filtrate esters were

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578 Blood Phosphorus. V

hydrolyzed with acid 24 hours, one aliquot of the solution ad- justed to pH 8.8, another to pH 7.4, and subjected to phosphatase action as in the original enzyme experiments. At pH 7.4 the hydrolysis was more extensive than at pH 8.8 but at either pH the hydrolysis proceeded 10 to 20 per cent faster after preliminary acid treatment. This was true despite the high amount of in- hibitory sodium sulfate which resulted from adjusting the pH.

/

HfXOSEDlPHOSHhTE /

TIME IN HOURS

CHART V. The hydrolysis of the phosphoric esters of blood filtrate, of blood phosphoglycerate, of p-glycerophosphate, and of hexosediphosphate with kidney phosphatase at 38” and at pH 7.4.

Phosphoglycerate was treated with acid for 24 hours and then with enzyme at pH 8.8 and 7.4. Similarly, hydrolysis proceeded faster at pH 7.4 than at pH 8.8 and more readily at either pH after preliminary acid treatment. The explanation for the increased rates of enzyme hydrolysis after preliminary hydrolysis with acid is not known. The acid may alter the compounds so that the enzyme can attack them more readily, or if the phos- phoglycerate is a diphospho ester, the enzyme and acid may act,

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E. Warweg and G. Stearns 579

selectively on the two phosphoric acid linkages. The conclusions to be drawn from an experiment of such nature are limited but the fact that both the isolated phosphoglycerate and the phosphoric ester in blood filtrate after heating with acid responded to enzyme activity more readily at pH 7.4 than at pH 8.8 supports the belief that they are identical compounds.

SUMMARY

Blood filtrate containing the acid-soluble phosphoric esters, phosphoglycerate isolated from blood, hexosediphosphate, and /3-glycerophosphate were subjected to acid and enzyme hydrolysis under the same conditions of temperature, salt content, trichloro- acetate ion and hydrogen ion concentration.

The hexosediphosphate was destroyed very rapidly and the @glycerophosphate very slowly by acid hydrolysis.

The acid hydrolysis of the phosphoric esters of the blood filtrate proceeded very rapidly for 10 minutes, progressively slower until the 12th hour, then at a slow but constant rate.

The velocity constant for the decomposition of the organic phosphorus in blood after 12 hours acid hydrolysis was the same as for phosphoglycerate isolated from blood, suggesting that the acid-resistant compound in the filtrate from human blood is identical with the phosphoglycerate isolated from pig blood. The reaction is shown to be monomolecular; therefore the phos- phoglycerate may be a monophospho ester or a diphospho ester in which both the phosphoric acid groups are split off at approxi- mately the same rate.

Further evidence of the identity of the isolated compound with that present in blood was shown by comparing the phosphatase hydrolysis of each. The phosphoglycerate isolated from pig blood was hydrolyzed by the enzyme with greater ease at pH 7.4 than at pH 8.8. The organic phosphorus of blood filtrate also was hydrolyzed more readily at pH 7.4 after the 24th hour. Before that time, the blood phosphoric esters were split more easily at pH 8.8.

Hydrolysis of hexosediphosphate and P-glycerophosphate oc- curred readily at either pH 7.4 or 8.8 but more rapidly in the more alkaline medium.

The per cent of organic phosphorus in the blood that exists

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Blood Phosphorus. V

as a phosphoglycerate can be determined by comparing the relative rates of acid hydrolysis of the organic phosphorus of the blood filtrate and the phosphoglycerate isolated from blood. Pyrophosphate has been shown to be hydrolyzed by acid within the first 10 minutes.

By calculation, the acid-soluble or ester phosphorus fraction of the blood of normal adults consists of about 68 per cent phos- phoglycerate phosphorus, with a range of 62 to 75 per cent, and approximately 11.4 per cent pyrophosphate phosphorus. The remainder may be a hexosephosphate.

This method of fractionation is offered as an aid in the deter- mination of gross changes in the phosphoric esters of whole blood during health and disease.

BIBLIOGRAPHY

1. Roche, J., Bull. Sot. chim. biol., 12, 636 (1930). 2. Lohmann, K., Biochem. Z., 202, 466 (1929). 3. Jackson, H., J. Biol. Chem., 69, 529 (1924). 4. Hoffman, W. S., J. Biol. Chem., 63, 675 (1925). 5. Buell, M. V., J. Biol. Chem., 108, 273 (1935). 6. Fiske, C. H., and Subbarow, Y., Science, 70, 381 (1929). 7. Lohmann, K., Biochem. Z., 233,460 (1931). 8. Mrockiewicz, U., Biochem. Z., 236, 267 (1931). 9. Greenwald, I., J. Biol. Chem., 63, 339 (1925).

10. J&t, H., 2. physiol. Chem., 166, 171 (1927). 11. Goodwin, H. W., and Robison, R., Biochem. J., 18, 1161 (1924). 12. Lawaczek, H., Biochem. Z., 146, 351 (1924). 13. Mai, H., 2. Kinder-he&, 46, 653 (1928). 14. Bakwin, H., Bodansky, O., and Turner, R., Proc. Sot. Exp. BioZ. and

Med., 29, 1228 (1932). 15. Barrenscheen, H. K., and Vasarhelyi, B., Biochem. Z., 230,330 (1931). 16. Meier, R., and Thoenes, IL, Arch. exp. Path. 7~. Pharmalcol., 161, 119

(1931). 17. Bomskov, C., 2. physiol. C’hem., 210, 67 (1932). 18. Kay, H. D., and Robison, R., Biochem. J., 18, 755 (1924). 19. Roche, J., Biochem. J., 26, 1724 (1931). 20. King, E. J., Biochem. J., 26, 1697 (1932). 21. Kay, H. D., Physiol. Rev., 12, 384 (1932). 22. Kobayashi, H., J. Biochem., Japan, 11, 173 (1929). 23. Bodansky, O., and Bakwin, H., J. BioZ. Chem., 104, 747 (1934). 24. Fiske, C. H., and Subbarow, Y., J. Biol. Chem., 66, 375 (1925). 25. Stearns, G., and Warweg, E., J. BioZ. Chem., 102, 749 (1933). 26. Kay, H. D., Biochem. J., 20, 790 (1926). 27. Davies, D. R., Biochem. J., 28, 529 (1934).

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28. Michaelis, L., J. Biol. Chem., 87, 33 (1930). 29. Erdtman, H., Z. physiol. Chem., 172, 182 (1927). 30. Jenner, H. D., and Kay, H. D., J. Biol. Chem., 93, 733 (1931). 31. Lawaczek, H., Klin. Woch., 4, 1958 (1925). 32. Asakawa, K., J. Biochem., Japan, 11, 143 (1929). 33. Asakawa, K., J. Biochem., Japan, 10, 157 (1928).

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Edna Warweg and Genevieve StearnsPHOSPHOGLYCERATE FRACTION

REFERENCE TO THE PHOSPHORUS, WITH PARTICULAR

THE ACID-SOLUBLE ORGANIC AND ENZYMATIC HYDROLYSIS OF

V. A COMPARATIVE STUDY OF ACID STUDIES OF PHOSPHORUS OF BLOOD:

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