.ANALYTICAL BIOCHEMISTRY 5, 542-547 (1963)

kleetrochrynatographic Separation of Inorganic Phos-

phate, Adenosine Monophosphate, Adenosine

Diphosphate, and Adenosine

Triphosphate ‘j2

T. R. SATO, J. F. THOMSON, AND W. F. DANFORTH

From the Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois

Received October 1, 1962

For the investigation of enzymically catalyzed phosphate-adenosine triphosphate exchange, a precise analytical procedure for the separation of phosphate, adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) was required. To avoid some of the disadvantages inherent in other methods, as reviewed by Klow- wen (1)) we have devised a sensitive and reliable electrochromatographic procedure. This method, which resembles those reported by Schaefer (2) and by von Korff (3), depends upon the differential electrical migration of the phosphorus compounds in the presence of zinc ions in a carefully buffered solution. In addition to improving the separability of the phos- phorus compounds, the zinc ions stabilize the ADP and the ATP against hydrolysis (4-S).

METHODS

Background or Buffer Solutions

An effective background or buffer solution, which determines the sep- arability of the several species, was selected by systematic variation of buffers, and of stabilizing salts as shown in the experimental section. The solution eventually chosen contained citric acid (0.035 M) plus sodium citrate (0.0148 M) as buffer, pH 3.8, and zinc acetate (0.003 M) to im- prove separability and stability of the phosphorus compounds. It satis- fied several requirements: effective separation of the phosphorus com-

’ This work performed under the auspices of the U. S. Atomic Energy Commission. *Taken from a thesis submitted by T. R. Sato to the Department of Biology,

Illinois Institute of Technology, in partial fulfillment of the requirements for the Doctor of Philosophy degree.

* Department of Biology, Illinois Institute of Technology, Chicago, Illinois.

542

ATP, ADP, AMP, AND PO4 SEPARATION 543

pounds, formation of well-defined zones, suitable pH and electrical conductivity, and noninterference with detection tests for the separated species.

Papers

The buffer solutions were stabilized with commercial filter paper made of wood pulp (Eaton-Dikeman Co., Grade 301, thickness 0.076 cm; Whatman filter papers No. 3 MM, No. 1, and No. 4). Several strips (2.54-cm width) were washed with 1 N nitric acid for one day and then with distilled water for four days. The washed strips were separated and then dried in air on clean sheets of polyethylene (7).

Materials

Nonradioactive AMP, ADP, and ATP were obtained from Pabst Laboratories. P”“-labeled ADP and ATP were prepared by eneymic exchange using rat liver mitochondria as described by Colowick and Kaplan (9). The AMP was not labeled by this method. H,P”?O, was obtained from Oak Ridge National Laboratories. The radioactivity served to locate the several species separated by electrochromatography. Mitochondria were prepared from rat liver according to the method of Schneider and Hogeboom (10) using 0.25 M sucrose.

Migration. Apparatus

Migrations at a moderate potential (about 5-10 volts/cm) were carried out in st,rips of the acid-washed paper 2.54 cm wide and 91.44 cm long. These strips were placed on a sheet of polyethylene across a water- cooled flat plate which was constructed by soldering copper tubing to one side of a copper plate 0.32 cm thick. For the migration studies, the strips of paper were first moistened with the buffer solutions. Aqueous solutions of the phosphorus compounds (about 5-10 ,~l of about 0.001 M “reaction mixture”) were placed in small zones near the cathode end of the moist paper. The strips of paper were then covered with poly- ethylene sheets, and direct current potential, either 5 volts/cm for 17 hr or lo-15 volts/cm for 2-4 hr, was applied. After the migration, the ends of the strips of paper were cut off, and the strips of paper were uncovered and allowed to dry in air.

PROCEDURE

For the isolation of the radioactive ADP and ATP formed by the exchange reaction, the electrochromatographic method was employed. The exchange reaction was carried out in 15-ml centrifuge tubes and the experiment was terminated by the addition of 0.1 vol of cold, 1 A!

544 SATO, THOMSON, AND DANFORTH

perchloric acid (or equivalent) with chilling. The precipitated protein was centrifuged, and the cold extract was neutralized immediately with 1 N potassium hydroxide to pH 6 to 7, using phenol red as an internal indicator. The mixture was chilled almost to the freezing point to bring about the precipitation of potassium perchlorate, which was removed by centrifugation. The supernatant solution was immediately submitted to electrical migration in the citrate-zinc acetate solution described above.

For detection and location of the radioactive compounds separated by the electrical migration, radioautographs were made with Kodak no screen X-ray film. Both the radioactive and the nonradioactive nucleo- tides, especially the AMP, could be detected with ultraviolet light by noting the dark spots on the fluorescing background of the paper. Phos- phoric acid was located by its blue color after treatment of the paper with molybdic acid and exposure to ultraviolet light (11). The quantity of Paa incorporated in the compounds on the electrochromatograms was measured with an automatic chromatogram scanner, Vanguard 880,” provided with a windowless, gas-flow tube and a precision rate counter, the output of which was taken to a recorder having a chart speed synchronized with that of the scanner mechanism. The strips were pulled under a slit of aperture 0.16 or 0.63 cm at rates ranging from 0.63 to 5.08 cm/min. Proper selection of conditions in this range made possible reproducible determinations of amounts of P3? in spots con- taining as few as 100 or as many as 2 X10” total counts per minute. The area under the curve for each radioactive compound was integrated by the automatic data system of the Vanguard 880. Each recorded peak was above the background level. Conversion to the quantity of P32 was effected by comparison with peaks for known standards of P32 com- pounds electrochromatographed and scanned at the same time (see legend for Fig. 2).

RESULTS AND DISCUSSION

The variation of the electrical migration rate of orthophosphat’e, AMP, ADP, and ATP in citric-citrate electrolyte with concentration of zinc acetate at pH 3.8 is illustrated by Fig. 1. As indicated above, sequence and separability were established with authentic nucleotide phosphorus compounds which were submitted to electrical migration and located by ultraviolet light.

Zones of ATP and orthophosphate migrated at the same rate in the absence of zinc ions. With optimum concentration of zinc ions (0.003 0.004 M), the ATP zone was well separated from the radioactive inor-

‘Vanguard Instrument Company, P. 0. Box 244, La Grange, Illinois.

ATP, ADP, AMP, AND PO1 SEPARATION 545

FIG. 1. Photograph of an ultraviolet contact print of electrochromatogram show- ing migration of the adenosine phosphate ions, AMP, ADP, ATP, and radioactive orthophosphate, H,P”‘OI in the citric acid-citrate electrolyte solution plus various concentrations of zinc ions. Each of the narrow paper strips contained 10 81 of radioactive phosphate plus nonradioactive AMP, ADP, and ATP; the background solution consisted of 0.035 M citric acid-O.0148 M citrate solution plus various con- centrations of zinc acetate adjusted to final pH 3.8. Zinc ion concentration: A, none; B, 0.001 A4 ; C. 0.003 M ; D, 0.004 M; E, 0.005 M; F, 0.01 M. Migration time, 4 hr; potential, 1000 volts/100 cm. The distance between letters A and E is 18 cm.

ganic phosphate zone, located by autoradiography. The rate of migration of ATP was determined primarily by the concentration of zinc ions; it decreased progressively with the addition of 0.001, 0.003, 0.004, 0.005, and 0.010 M zinc ions. The addition of the zinc ions to the citric-citrate electrolyte had only a slight effect on the rate of migration of AMP, ADP, and orthophosphate. For effective separations, a critical concen- tration of zinc salt is required to cause the ATP to migrate more slowly than phosphate but faster than ADP.

The effect of varying the pH of the background electrolyte from pH 3.0 to 5.5 is illustrated in Fig. 2. In these experiments the zinc ion concentration was constant at 0.003 iM. A pH of 3.0 (A, Fig. 2) pro- vided a separat,ion of ADP plus ATP from PO,, but no separation of ADP and ATP. A pH of 3.8 (B, Fig. 2) provided an excellent separation of the four species. At pH values of 4.5 and greater (C, D, E, Fig. 2), there was excellent separation of ADP and ATP but no separation of ATP and PO,. The most effective separations are obtained at pH 3.8.

The definition of the zones in the electrochromatogram is indicated by the radioautograph reproduced as Fig. 2. The sharpness of the zones in a chromatogram made under favorable conditions (B, Fig. 2) was confirmed by scanning the paper with a strip counter constructed by Kisieleski and Smetana (12) as shown by the tracing in Fig. 3.

546 SATO, THOMSON, AND DANFORTH

FIG. 2. Effect of pH upon separation of AMP, ADP, ATP, and phosphate ions (PO,) from the ATP-PO, exchange reaction mixture. Migration in 0.035 M citric acid-O.01458 M sodium citrate electrolyte plus 0.003 M zinc acetate. pH values: A, 3.0; B, 3.8; C, 4.5; D, 5.0; E, 5.5. Each zone contained 10 ~1 of a solution of radioactive ADP, ATP, and PO, and nonradioactive AMP. ADP added, 154 cps; found, 151. ATP added, 212 cps; found, 208. PO, added, 116 cps; found, 112. Poten- tial, 1060 volts/80 cm; migration time, 4 hr. The distance between the letters A and E is 18 cm.

200-

P F In

E a

0 IOO-

B*

I I I I I 0 IO 20 30 40

cm

FIG. 3. Curve representing distribution of radioactive species in an electro- chromatogram formed by migration of the ATP-PO, exchange reaction mixture at pH 3.8. The electrochromatogram was the same one that provided radioautograph B shown in Fig. 2. See text for methods.

ATP, ADP, AMP, AND PO4 SEPARATION 547

The results summarized in Figs. l-3 show that electrochromatography under carefully controlled conditions is an effective technique for the separation of mixtures of H,PO,, AMP, ADP, and ATP. Since separa- tions were equally good with pure compounds or with mitochondrial extracts, the met,hod should be suitable for studies on biological material.

SUMMARY

Electrochromatography with carefully adjusted electrolyte solutions (0.003 M zinc acetate, 0.035 M citric acid, and 0.0148 A4 sodium citrate’) at pH 3.8 is an effective t’echnique for the separation of mixtures of phosphoric acid, adenosine monophosphate, adenosine diphosphate, and adenosinc triphosphatc.

ACKNOWLEDGMENT

We wish to express our gratitude to Dr. H. H. Strain for reading this manuscript and for his comments.

REFEREXCES

1. KLOwWEN, H. M., J. Chromatog. 7, 216 (1962). 2. SCHAEFER, J., Arch. Exptl. Pathol. Pharmacol. 236, 48.3 (1959). 3. VON KORFF, R. W., Anal. Biochem. 3, 244 (1962). 4. BELL, R. N., Ind. Eng. Chem., Anal. Ed. 19, 97 (1947). 5. BRITZKE, E. V., AND DRAGUNOV, S. S., J. Chem. Ind. (U.8S.R.) 4, 49 (1927). 6. GERBER, A. B., AND MILES, F. T., Ind. Eng. Chem., An&. Ed. 10, 519 (1958). 7. SATO, T. R., Anal. Chem. 31, 841 (1959). 8. STRAIN, H. H., .4ND SAW, T. R., U. S. Atomic Energy Comm. TID-7512 1956,

pp. 175-182. 9. COLOWICK, S. P., AND KAPLAN, N. O., in “Methods in Enzymology” (S. P. Cola-

wick and N. 0. Kaplan, eds.), Vol. IV, p. 853. Academic Press, New York, 1957.

10. SCHNEIDER, W. C., AND HOGEBOOM, G. H., J. Biol. Chem. 183, 123 (1950). 11. BANDURSKI, R. S., AND AXELROD, B., J. BioZ. Chem. 193, 495 (1951). 12. KISIELESKI, W. E., AND SMETANA, F., Atompraxis 4, 261 (19%).