the objective of the present work was to provide a means for separating and indentifying phosphate esters involved in glycolysis in higher plants. Paper chromatography of phosphate esters has been employed by several workers, most notably Benson et al. (1) and Hanes and Isherwood (2). Benson's procedures were not primarily designed for identification of phosphate esters and gave low Rr values for the phosphate compounds of particular interest to us. The unidimensional methods of Hanes and Isherwood do not result in adequate resolution of the complex mixtures such as are obtained from our plant materials. 



The present procedure is based on two-dimensional chromatography with successive development in an acid and in a basic solvent. The solvents finally selected gave the best over-all resolution of the intermediates involved in plant glycolysis. Undoubtedly the resolution of certain pairs of compounds may be improved by suitable modifications. We have in addition made certain improvements in the procedure for locating the chromatographed materials

Axelrod, Bernard

Bandurski, Robert S.

English

Caltech Authors - Main

THE CHROMATOGRAPHIC IDENTIFICATION OF SOME 
BIOLOGICALLY HMPORTANT PHOSPHATE ESTERS* 
BY ROBERT S. BANDURSKI 
(From the Kerckhoff Laboratories of Biology, California Institute of Technology, 
Pasadena, California) 
AND BERNARD AXELRODt 
(From the Enzyme Research Division, Bureau of Agm’cultural and Industrial Chemistry, 
Agricultural Research Administration, United States Department of Agriculture, 
Albany, California) 
(Received for publication, June 29, 1951) 
The objective of the present work was to provide a means for separating 
and identifying phosphate esters involved in glycolysis in higher plants. 
Paper chromatography of phosphate esters has been employed by several 
workers, most notably Benson et at. (1) and Hanes and Isherwood (2). 
Benson’s procedures were not primarily designed for the identification of 
phosphate esters and gave low Rp values for the phosphate compounds of 
particular interest to us. The unidimensional methods of Hanes and 
Isherwood do not result in adequate resolution of the complex mixtures 
such as are obtained from our plant materials. 
The present procedure is based on two-dimensional chromatography 
with successive development in an acid and in a basic solvent. The sol- 
vents finally selected gave the best over-all resolution of the intermediates 
involved in plant glycolysis. Undoubtedly the resolution of certain pairs 
of compounds may be improved by suitable modifications. We have in 
addition made certain improvements in the procedure for locating the 
chromatographed materials. 
EXPERIMENTAL 
Reagents and Materials- 
Acid solve&. Methanol 80 volumes, formic acid 15 volumes (analysis 
88 per cent by weight), and HzO, 5 volumes. 
Basic solvent. Methanol 60 volumes, ammonium hydroxide 10 volumes 
(sp. gr. 0.9015), and Hz0 30 volumes. 
Spray reagent. Hanes-Isherwood reagent (2). 5 ml. of 60 per cent 
* Enzyme Research Division Contribution No. 140. Report of work supported 
in part by the Herman G. Frasch Foundation. 
t Present address, Kerckhoff Laboratories of Biology, California Institute of 
Technology, Pasadena, California. 
495 
406 PHOSPHATE ESTERS 
(weight by weight) perchloric acid, 25 ml. of 4 per cent (weight by volume) 
ammonium molybdate, 10 ml. of N HCl, and 60 ml. of HzO. 
Paper. Schleicher and Schuell, No. 589, blue ribbon filter paper 28 
cm. square was found satisfactory for our purpose.’ Even analytical filter 
papers contain sufficient inorganic phosphate to give a visible band on de- 
velopment and spraying. The color is very light but there may be suffi- 
cient phosphate accumulated at the intersection of the bands produced in 
the acid and basic solvents to produce a spurious spot. If desired, this 
difficulty may be eliminated by washing the paper with the acid solvent 
before use. 
Development of Chromatogram 
Chromatography of phosphate esters is best accomplished with the free 
acids. Salts of the esters are readily converted to the corresponding acid 
and the metallic ions removed by shaking solutions or suspensions of the 
salts with a cation exchanger such as Dowex-50. A suitable quantity of 
solution containing the decationized compounds to be chromatographed 
is applied to one corner of the filter paper about 5 cm. from the bottom and 
side edge. It has been found desirable to limit the size of the spot to 
approximately 0.5 cm. in diameter. In practice this amounts to appli- 
cations of 2 ~1. Since a minimum of 0.1 y of P is generally needed for 
detection, it may be necessary to make multiple applications with dilute 
solutions, in which case the spot is air-dried each time. The paper is then 
formed into a cylinder and stapled together, avoiding contact of the ad- 
jacent edges. A cylindrical jar 15 cm. in diameter and 30 cm. tall, covered 
by a glass square and containing 100 ml. of the solvent, serves as a chro- 
matographic chamber. A soft iron wire is inserted diametrically through 
the upper end of the paper cylinder, which is then suspended over the 
solvent by means of a magnet placed on top of the glass cover. We have 
found it advisable to use the acid solvent first and to conduct equilibra- 
tion and development at 2”. After a 2 hour equilibration, the paper cyl- 
inder is dropped into the solvent by removing the magnet (3). The 
chromatogram is developed until the solvent front reaches the upper edge 
ofthe paper. LkaZZy 6 to 6.5 Illours are requiied. The chromatogram i5 
then thoroughly dried in front of a fan at room temperature. The paper 
cylinder is next reformed so that the second development will be at right 
angles to the first. The equilibration procedure is repeated with the alka- 
line solvent. Development in the alkaline solvent requires 12 to 15 hours. 
1 The mention of special instruments or materials throughout this paper does not 
imply that they are endorsed or recommended by the Department of Agriculture 
over others of a similar nature not mentioned. 
R. S. BANDURSKI AND B. AXELROD 407 
Detection of Compounds 
The paper is air-dried and then examined by appropriate methods. 
Carter (4) has shown that certain nucleotides may be detected on paper 
when viewed with short ultraviolet radiation (2536 A). This suffices to 
locate adenosinetriphosphate (ATP), adenosinediphosphate (ADP), and 
adenosinemonophosphate (AMP). 
The spray reagent used for the visualization of phosphorylated com- 
pounds is that of Hanes and Isherwood (2). The procedure for developing 
the color and hydrolyzing the esters has been modified, since, in our hands, 
their procedure does not serve to locate such difficultly hydrolyzable esters 
as phosphoglyceric acid. After the paper is sprayed at the rate of 1 to 2 
ml. per 100 sq. cm., inorganic phosphate appears as a yellow spot. The 
paper is dried at 85” for 1 minute, at which time glucose-l-phosphate is 
revealed by a yellow to blue color. The paper is then exposed to ultra- 
violet radiation at a distance of about 10 cm. from a General Electric 
germicidal lamp (rated at 25 microwatts of 2537 A radiation per sq. cm. 
at 1 meter) for 10 minutes. All of the organic phosphate compounds will 
now appear as blue spots, whereas inorganic phosphate produces a yellow- 
green color. An alternate method for developing the color consists of 
heating the paper for 5 minutes at 85” and autoclaving for 2 minutes at 
8 to 10 pounds. The paper is now uniformly blue in color. When the chro- 
matogram is exposed to ammonia vapors, the blue color of the background 
disappears, while the color due to phosphomolybdate blue remains. 
Results 
Table I gives the Ra values of some of the phosphate esters of glycolysis 
as well as ortho-, pyro-, and tripolyphosphates in the acid and alkaline 
solvent. Fig. 1 illustrates the two-dimensional chromatographic separa- 
tion of a mixture of inorganic phosphate, ATP, fructose-1,8diphosphate, 
3-phosphoglyceric acid (3-PGA), and 2-phosphoglyceric acid. These com- 
pounds are normally found in the “barium-insoluble fraction,” prepared 
as described by Umbreit, Burris, and Stauffer (5). Fig. 2 shows the be- 
havior of a mixture of some substances occurring in the “barium-soluble 
fraction” (5), namely glucose-l-phosphate, glucose-6-phosphate, fructose- 
6-phosphate, 3-AMP, and phosphoenolpyruvic acid. 
was included as a marker. 
Inorganic phosphate 
In our experience, the position of the spots 
relative to each other has proved more useful than Rp values. This is 
particularly true when complex impure mixtures are chromatographed (1). 
The Rr values, while fairly reproducible with a single batch of paper, are 
not the same for different grades of papers and, further, are affected by 
washing of papers. 
408 PHOSPHATE ESTERS 
TABLE I 
RI Values of Phosphate Esters 
Compound 
-r 
Acid solvent Alkaline solvent 
-- -- 
Adenosinetriphosphate. .................... 0.12 0.15 
Adenosine-3-phosphate. ................... 0.17 0.32 
Glucose-l-phosphate ....................... 0.27 0.60 
Fructose-6-phosphate. ..................... 0.34 0.44 
Tripolyphosphate .......................... 0.37 0.06 
Glucose-6-phosphate. ...................... 0.38 0.48 
Fructose-l ,6-diphosphate .................. 0.40 0.24 
2-Phosphoglycerate. ....................... 0.46 0.18 
Pyrophosphate ............................ 0.46 0.05 
3,-Phosphoglycerate. ....................... 0.50 0.35 
Phosphoenolpyruvate. ..................... 0.52 0.46 
Orthophosphate ........................... 0.63 0.28 
Rf ALKALINE SOLVENT 
- 
RF 
- 
0 
3 
0‘ 
FIG. 1 FIG 2 
FIG. 1. Two-dimensional chromatogram of glycolytic intermediates. “Barium- 
insoluble fraction:” 1, ATP; d, orthophosphate; 5, fructose-1,6-diphosphate; 4, 3- 
phosphoglyceric acid; 6, 2-phosphoglyceric acid. 
FIN. 2. Two-dimensional chromatogram of glycolytic intermediates. “Barium- 
soluble fraction:” 9, orthophosphate; 6, adenosine-3-phosphate; 7, phosphoesol- 
pyruvic acid; 8, glucose-l-phosphate; 9, glucose-g-phosphate; 10, fructose-6-phos- 
phate. 
The identification of unknown constituents in mixtures is greatly facili- 
tated when either the unknown compounds or known authentic samples 
are available with radioactive labels. After chromatographing a mixture 
of the unknown and known compounds, the exact coincidence of position 
R. S. BANDURSKI AND B. AXELROD 409 
and shape of the spots on the chromatogram and the radiogram makes the 
identification of the unknown compounds highly probable. 
The technique described above has found application in the study of 
the phosphorylated compounds formed by the incorporation of radioactive 
phosphate by pea seed meal, pea seed extract (6), and orange flavedo 
extract.2 
DISCUSSION 
Selection of Solvents 
The selection of the solvents was based on the results of numerous tests 
carried out by the method of Rockland and Dunn (7) by employing small 
filter paper sectors developed in test-tubes. It was thus possible to survey 
quickly several hundred solvent combinations for their ability to resolve a 
mixture of glucose-l-phosphate, 3-PGA, and inorganic phosphate. The 
following solvents were tested singly or in certain combinations: water, 
methanol, ethanol, normal, and isopropanol, normal, secondary, tertiary, 
and isobutanol, isopentanol, furfuryl alcohol, glycol monomethyl ether, 
diethyl ether, isopropyl ether, dioxane, acetonitrile, acetone, diethyl car- 
bonate, ethyl acetate, benzylamine, ethylenediamine, piperidine, pyridine, 
ammonium hydroxide, hydrochloric, formic, and glacial acetic acids. In 
summary these generalizations may be made. In the absence of water no 
movement occurred except with methanol, ethanol, glycol monomethyl 
ether, and acetonitrile. The Rp values for the esters in these solvents 
could be enhanced by the addition of water. However, a high concen- 
tration of water caused high RF values and poor separation of the esters. 
Even with optimum amounts of water, poor resolution and broadly spread 
spots were obtained over the pH interval of 4 to 9. This difficulty was 
alleviated by the incorporation of acids or bases. Lugg and Overell (8) 
encountered a similar difficulty in the chromatography of organic acids, 
which they overcame by addition of “swamping” concentrations of a strong 
acid. The solvents which Hanes and Isherwood (2) found most suitable 
for the chromatography of phosphate esters were either strongly acidic or 
basic. In the present work the choice of acid or base, as of any solvent, 
was based first on the necessity for easy removal. Thus formic and hy- 
drochloric acids were suitable from this point of view, but the use of hydro- 
chloric acid resulted in excessive damage to the paper. 
:)-The order of the chromatographed compounds relative to one another 
was the same for all acid solvents used. Other orders are, however, ob- 
tained by the use of alkaline solvents, as was evident from the work of 
Hanes and Isherwood. Of all the bases listed, only NH,OH and piperidine 
were found to be useful; the former was selected because of its lower cost 
* Axelrod, B., and Bandurski, R. S., unpublished. 
410 PHOSPHATE ESTERS 
and greater volatility. Methanol, ethanol, n-propanol, acetonitrile, glycol 
monomethyl ether, and dioxane were all suitable as the neutral component 
of the solvent. Methanol was selected because it caused more rapid de- 
velopment than the other solvents named. The composition of the mix- 
tures finally chosen was based upon experiments in which the proportions 
of the components were varied in small steps. 
Conjirmatory Identijication of Phosphate Spots 
The characteristic properties by which glucose-l-Pod, inorganic phos- 
phate, and the purine derivatives may be detected have already been dis- 
cussed. These substances, if present, can serve as index compounds for 
the location of other esters. The various carbohydrate esters may be 
located by the use of specific carbohydrate sprays (9-11). It is sometimes 
useful to add one or more authentic phosphate esters in large amounts to 
obtain index spots. It is further possible to identify an unknown com- 
pound with a known by loading a second chromatogram with the known 
material and observing the augmentation of the spot. 
SUMMARY 
1. A two-dimensional chromatographic method for the identification of 
some phosphate esters of biological importance, including inorganic poly- 
phosphates, is described. 
2. An acid solvent consisting of methanol, formic acid, and water and a 
basic solvent containing methanol, ammonia, and water are used. 
3. An improved method of color development involving ultraviolet light 
permits the hydrolysis of resistant esters and the minimization of back- 
ground color. 
It is a pleasure to acknowledge the cooperation of Mr. George Ellman 
and the assistance of Miss Rosamond S. Baker in this work. 
BIBLIOGRAPHY 
1. Benson, A. A., Bassham, J. A., Calvin, M., Goodale, T. C., Haas, V. A., and 
Stepka, W., J. Am. Chem. Sot., 72, 1710 (1950). 
2. Hanes, C. S., and Isherwood, F. A., Nature, 164,1107 (1949). 
3. Zarudnaya, K. I., Thesis, University of Missouri (1950). 
4. Carter, C. E., J. Am. Chem. Sot., 72,1466 (1950). 
5. Umbreit, W. W., Burris, R. H., and Stauffer, J. F., Manometric techniques and 
tissue metabolism, Minneapolis, 2nd edition (1950). 
6. Axelrod, B., Bandurski, R. S., and Saltman, P., Federation Proc., 10, 158 (1951). 
7. Rockland, L. B., and Dunn, M. S., Science, 111, 332 (1950). 
8. Lugg, J. W. H., and Overell, B. T., Australian J. SC. Res., Series A, 1, 98 (1948). 
9. Partridge, S. M., Biochem. J., 42, 238 (1948). 
10. Partridge, S. M., Nature, 164, 443 (1949). 
11. Forsyth, W. G. C., Nature, 161, 239 (1948). 


The chromatographic identification of some biologically important phosphate esters

Manometric techniques and tissue metabolism, Minneapolis, 2nd edition

Thesis,

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The chromatographic identification of some biologically important phosphate esters

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