ANALYTICAL BIOCHEMISTRY 254, 249–253 (1997)ARTICLE NO. AB972437

Determination of Inorganic Phosphate by CouplingThymidine Phosphorylase and Reversed-Phase High-Performance Liquid Chromatography: Applicationto Tonoplast Pyrophosphatase Activity

Max Hill1 and Bernard ArrioERS 571 du CNRS, Bioenergetique Membranaire, Bat. 432, Universite de Paris Sud,Centre d’Orsay, 91405 Orsay Cedex, France

Received June 17, 1997

various substances compete for free molybdate. Nu-We developed an HPLC method for the measurement merous modifications of the original Fiske and Sub-

of inorganic phosphate using thymidine phosphoryl- barow procedure have been published (8–13) to correctase (EC 2.4.2.4). This enzyme catalyzes the phosphorol- some of these problems, but none solve all of them.ysis of thymidine to 2-deoxyribose 1-phosphate and We have developed a new method based on an en-thymine. Thymine release was measured at 265 nm zyme reaction and HPLC separation and quantifica-after separation by reverse-phase HPLC. The assay tion. In previous work, we described an HPLC tech-was sensitive enough to detect as little as 10 pmol of nique to follow the simultaneous measurement ofPi. The response to the phosphate concentration was changes in adenylic nucleotides during ATP hydrolysislinear from 1 to 100 mM. The value of this method was (14, 15). This technique was applied to the 5*-nucleoti-demonstrated in an analysis of the kinetics of Pi re- dase activities of the tonoplast. The main advantagelease from PPi in the presence of Catharanthus roseus of this quantitative technique is that it differentiatestonoplast pyrophosphatase. q 1997 Academic Press

between nucleotidase and phosphotransferase activi-ties. However, the technique was unsuitable for Pi ti-tration.

We have therefore used thymidine phosphorylaseVarious assays have been described for measuringfrom Escherichia coli for quantitative titration of Pi .inorganic phosphate released from nucleotides and py-This enzyme, first described by Manson and Lampenrophosphate hydrolyzed by phosphohydrolases. These(16), catalyzes the following reaction: thymidine /methods involve either the extraction of 32Pi releasedphosphate } thymine / 2-deoxyribose 1-phosphate.(1, 2), or the use of coupled enzyme reactions with fluo-After quenching the reaction, thymine can be quanti-rescent (3) or UV2 absorbing substrates (4–6).fied by a fast HPLC separation and compared with aColorimetric titration, first developed by Fiske andPi standard curve. We illustrate our method with aSubbarow (7), is one of the most widely used spectro-study of pyrophosphatase activity in Catharanthus ro-photometric assays for the determination of inorganicseus tonoplast.phosphate. It is based on the quantification of Pi as a

In the present paper we describe this sensitive andcolored complex of molybdophosphoric acid or molybdo-specific method for the determination of phosphate.vanaphosphoric acid. Although the method is sensitive

and simple, it has drawbacks; the color intensity istime dependent, labile substrates are hydrolyzed, and MATERIALS AND METHODS

Reagents1 To whom correspondence should be addressed. Fax: 01 69 8537 15. Thymidine phosphorylase from E. coli (Product No.2 Abbreviations used: HPLC, high-performance liquid chromatog- T2807), thymidine, and thymine were obtained fromraphy; UV, ultraviolet; Pi , inorganic phosphate; PPi, inorganic pyro-

Sigma (St. Louis, MO). Tetrasodium pyrophosphatephosphate; Tris, tris(hydroxymethyl)aminomethane; PCA, perchloricacid; BCA, bicinchoninic acid. was purchased from Fluka (Buchs, Switzerland). All

2490003-2697/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

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other chemicals and solvents were obtained from were incubated in 890 ml of 50 mM Tris–HCl buffer,pH 7.3, containing 50 mM KCl, 5 mM MgSO4, 0.15 mMMerck (Darmstadt, Germany).NH4 heptamolybdate, 100 mM thymidine, and 5 mMKF, when this inhibitor was used. The reaction wasPreparation of the Vesiclesinitiated by adding, under magnetic stirring at 207C,Tonoplast-enriched vesicles were prepared and char- 100 ml of 10 mM Na4O7P2 stock solution. At determinedacterized as previously described (17, 18) with some time intervals, aliquots of 100 ml of the reaction mix-modifications. A tonoplast suspension of 500 ml was ture were withdrawn and incubated at 987C for 1 mincentrifuged at 100,000g for 30 min. The resulting pellet in tubes in a heating block. After centrifugation for 2was resuspended in 1500 ml of 100 mM Tris–HCl min at 14,000g the supernatant was analyzed by HPLCbuffer, pH 7.3, then centrifuged as above. The new pel- or stored at 47C. Ten kinetics could be run at the samelet was resuspended in 700 ml of the same buffer, then time. Control experiments were carried out with to-stored at 27C and used immediately for activity mea- noplast vesicles; PPi and KF blanks were subtracted.surements. H/-PPase hydrolytic activity was measured as the lib-eration of free Pi (divided by 2) from PPi.Standard Solutions

To minimize background absorbance and Pi contami- HPLC Techniquenation, we used plastic ware and demineralized then Thymine and thymidine were separated by HPLCdistilled water. Thymidine phosphorylase (800 ml, 552 reversed-phase chromatography with isocratic elution.U) was dialyzed for 15 h against 2 liters of 10 mM Tris– The mobile phase was a mixture of 9% MeOH and 91%HCl buffer, pH 7.3, then diluted to obtain a solution 0.1 M KH2PO4 buffer, pH 7, and the flow rate was 0.9containing 100 U per milliliter. The solution was di- ml/min. All the solutions were filtered and degassed.vided and samples were frozen at 0207C until use. The volume injected was 10 ml. Detection was per-For calibration, thymine standard solutions were formed at 265 nm. Thymine was quantified by mea-prepared from a 0.25 mM thymine stock solution. Thy- surement of peak areas using an external multilevelmine and thymidine concentrations were measured calibration method. The amount of thymine was evalu-spectrophotometrically: thymine at 264.5 nm emax ated by comparison with samples containing known(11003) Å 7.9 (pH 7), and thymidine at 267 nm emax amounts of thymine. All operations, sample injections,(11003) Å 9.7 (pH 7). Pi standard solutions were ob- and isocratic elutions, UV detection, peak integration,tained by dilution of a 1 mM NaH2PO4 stock solution. and column regeneration were automatically controlledBefore use NaH2PO4 was dried at 807C overnight. Stan- by HPLC system manager software; the system oper-dard solutions were stored at 07C. ated under full automation from sample injection to

data report.Pi Calibration Curve

Samples containing 0.1–10 mM of thymidine, 0–100 EquipmentmM of Na2HPO4 in 950 ml of 10 mM Tris–HCl buffer, The HPLC system consisted of two Model 303 pumpspH 7.3, were placed into 1.5-ml capped microcentrifuge and a Model 231 autosamper from Gilson (Villiers letubes; the reaction was initiated by adding 5 U of thy- Bel, France). Peak monitoring was performed with amidine phosphorylase in 50 ml of the precedent buffer. Model 165 variable-wavelength UV detector from Beck-At time intervals (from 5 to 55 min) the reaction was man. Thymine and thymidine were analyzed on a Su-stopped using method (a) or (b). (a) One hundred micro- pelcosil LC-18-DB column (3 mm, 4.6 1 33 mm) fromliters of the reaction mixture was withdrawn and incu- Supelco, protected by a Brownlee RP 18 (7 mm, 3.2 1bated at 987C for 1 min in tubes in a heating block. 15 mm) guard column.The tubes were centrifuged for 2 min at 14,000g andthe supernatants were analyzed by HPLC or stored at

Protein Concentrations47C. (b) One hundred microliters of the reaction mix-ture was withdrawn and mixed with 50 ml of 10% PCA. Protein concentrations were determined by the bicin-

choninic acid (BCA) method (19) using the micro-BCASince nucleotides are not stable at acid pH, after cen-trifugation for 2 min at 14,000g, 100 ml of supernatant protein assay reagent kit from Pierce.was adjusted to pH 7 with 100 ml of 0.5 M Na2HPO4

buffer, pH 8, and analyzed by HPLC. RESULTS AND DISCUSSION

HPLC allows separation, identification, and quanti-Measurement of H/-PPase Activity tation of nucleotides and their degradation products

other than Pi. HPLC is a very valuable tool for measur-In a typical assay, 10 ml of the tonoplast suspension(39 mg of protein) and 5 U of thymidine phosphorylase ing nucleotidases activities (14, 15, 20, 21). The main

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CHROMATOGRAPHIC DETERMINATION OF INORGANIC PHOSPHATE 251

advantage of this technique, applied to membranes,is to provide information about unexpected secondaryreactions and to reveal new enzymatic activities. Thishas been already illustrated on tonoplast vesicles fromC. roseus (14, 15). A pyrophosphatase activity has beencharacterized on these vesicles (18, 22). Pyrophosphatehydrolyzing activities are usually assayed by measur-ing the release of Pi with the colorimetric method ofFiske and Subbarow (7). However, the absorbance ofboth the controls and the samples increased with timeto an unacceptable level, such that it was impossible tostudy simultaneously several kinetics and accumulatesamples. In addition, different inhibitors of the to-noplast enzymes (vanadate, nitrate) and NH4 heptamo-lybdate, preventing eventual phosphatase contamina-tion, interfere with the titration reagents. Coupledenzyme assays have been described for measuring inor-ganic phosphate; the limitations of these methods arethe lack of sensitivity (below 1 mM) (6) and the instabil-ity of the commercially unavailable substrate (5). Onthe other hand, all these fluorimetric or spectrophoto-metric methods (3, 5) could not be used directly withoutmodifications in the presence of membrane suspensionsand when substrates absorbing in the same spectralrange are used. A two-coupled enzyme assay involvingan oxidoreduction step interfered with a membranecontaining electron transfer systems (6).

Therefore, a single enzyme assay is preferable to pre-vent these difficulties. We chose the phosphorolytic ac-tivity of thymidine phosphorylase from E. coli (23). Thisenzyme catalyses the reaction thymidine / phosphate} thymine/ 2 *-deoxyribose 1-phosphate, which allowsthe Pi titration by thymine measurement. This low-costenzyme is specific for pyrimidine 2 *-deoxyribosides,and consequently this specificity eliminates artifactswith purine compounds, particularly with adenine nu-cleotides involved in the energization process of vacuolemembrane. Conventional assays using spectrophoto-metric measurement of the thymine produced are notsensitive enough, because of the small difference be-tween the molar extinction coefficients of thymidineand thymine.

Therefore, the first step was to separate thymidine andthymine by HPLC. A short column containing 3-mm-di- FIG. 1. HPLC titrations of Pi standards in the presence of (l) 10ameter particles was chosen. Isocratic separation was mM thymidine. (.) Thymine measured: a, for 1 mM Pi; b, for 100

mM Pi. Chromatographic conditions: Supelcosil LC-18-DB column (3optimized to obtain the best resolution with the minimalmm, 4.6 1 33 mm) protected by a Brownlee RP18 (7 mm, 3.2 1 1.5analysis time. Figure 1 shows a typical chromatogrammm) guard column. Injection volume, 10 ml. Solvent system: 0.1 Mafter reaction with thymidine phosphorylase. The method KH2PO4 buffer, pH 7, 9% MeOH (v/v); isocratic elution, flow rate,

is characterized by an excellent reproducibility and a 0.9 ml/min.short analysis time (3.5 min). Moreover, isocratic elutionallowed injection of a new sample immediately after thy-midine elution. Series of 80 to 100 samples were routinely Linearity of the function peak area versus thymine con-

centration was verified over the range 2.5 to 100 mM (seeanalyzed without column regeneration. The peaks weremonitored at 265 nm to detect thymine and thymidine. Fig. 2). It was linear with a correlation coefficient of 0.999.

The thymine detection limit was lower than 10 pmol forThymine concentrations were calculated from the peakareas using an external multilevel calibration method. a 10-ml injection volume.

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HILL AND ARRIO252

FIG. 4. Standard calibration curve for Pi titration by thymine inthe presence of 10 mM thymidine. For experimental conditions seeFIG. 2. Linear relationship between thymine peak areas and theMaterials and Methods. Chromatographic conditions as in Fig. 1.concentrations of thymine injected. Each point is the average of five

replicates from a single experiment. Chromatographic conditions asin Fig. 1.

centration was adjusted so that the reaction was com-plete in less than 5 min; after this time, the thymineconcentration remained stable. This avoided theThe second step was the setting of the enzymaticquenching of the reaction and allowed analysis of aassay. In this reaction the Michaelis constants for thy-large series of samples. As the enzyme may alter themidine and phosphate are 0.38 and 0.35 mM, respec-HPLC column and the elution patterns, it was elimi-tively (23). Using a saturation concentration of 10 mMnated by a thermal denaturation at 987C, followed bythymidine, the reaction can be considered a bimolecu-centrifugation or filtration of the samples.lar reaction between phosphate and a complex of thy-

The optimum pH for thymidine phosphorolysis is 6.3midine with the enzyme. As shown in Fig. 3, from 0 to(23). Under our experimental conditions (see Materials100 mM phosphate, the most extended linearity be-and Methods), there were no differences in the thyminetween phosphate concentration and thymine formationproduction at pH between 6 and 7.5 (results notwas observed with this 10 mM thymidine concentra-shown). To obtain the Pi calibration curve, assays weretion.performed at pH 7.3 in the presence of 10 mM thymi-The two products of the reaction (thymine and 2 *-dine and Pi standard solutions.deoxyribose 1-phosphate) are considered as inhibitors

The reaction was initiated by adding thymidine phos-of the reaction (23–26). Moreover, according to Iltzschphorylase (5 units/ml). After thermal denaturation thy-et al. (25), product inhibition was avoided when themine was assayed by HPLC, or samples could be storedenzyme was saturated by thymidine. The enzyme con-at 47C for several weeks. The relationship between Pi

and thymine concentrations is shown in Fig. 4. Theresponse was linear with phosphate concentrationsfrom 1 to 100 mM. Under these conditions, Pi/thyminestoichiometry was 1.

Tonoplast pyrophosphatases have been extensivelystudied (27–32). Pyrophosphatase activity in C. roseustonoplast was previously characterized (18, 22); weused this membrane to validate our method. Kineticsstudies were carried out as described under Materialsand Methods. Figure 5 shows the hydrolysis of PPi bypyrophosphatase. In the presence of 5 mM KF, the ac-tivity was completely inhibited. These results agreewith those of the literature (33–35).

In summary, the new method described here can beused for Pi determination with a sensitivity lower than10 pmol and a linear response in a range from 1 to 100

FIG. 3. Effect of various thymidine concentrations on thymine pro-mM. The method is suitable for measuring inorganicduced as a function of Pi . (j) 0.1 mM thymidine, (l) 0.2 mM thymi-phosphate and the kinetics of Pi release from pyrophos-dine, (h) 1 mM thymidine, (s) 2 mM thymidine, (n) 10 mM thymi-

dine. Chromatographic conditions as in Fig. 1. phatases.

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CHROMATOGRAPHIC DETERMINATION OF INORGANIC PHOSPHATE 253

13. Kagami, O., and Kamiya, R. (1995) Methods Cell Biol. 47, 147–150.

14. Hill, M., Dupaix, A., Volfin, P., Kurkdjian, A., and Arrio, B.(1987) Methods Enzymol. 148, 132–142.

15. Hill, M., and Arrio, B. (1995) I. J. B. C. 1, 177–190.16. Manson, L. A., and Lampen, J. O. (1951) J. Biol. Chem. 193,

539–547.17. Dupaix, A., Johannin, G., and Arrio, B. (1989) FEBS Lett. 249,

13–16.18. Dupaix, A., Guyen, L., Hill, M., and Arrio, B. (1990) Arch. Bio-

chem. Biophys. 278(2), 392–397.19. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K.,

Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke,N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem.150, 76–85.

20. Schmidt, A. L., and Briskin, D. P. (1993) Arch. Biochem. Biophys.FIG. 5. H/-PPase activity of Catharanthus roseus tonoplast vesi-306, 407–414.cles, (j) with or (h) without 5 mM KF. Initial substrate concentra-

tion: 1 mM PPi; tonoplast from C. roseus: 39 mg/ml; incubation me- 21. Naud, I., Brugidou, C., Amalou, Z., and Marin, B. (1991) CRdium, see measurement of H/-PPase activity under Materials and Acad. Sci. Paris III, 273–279.Methods. Chromatographic conditions as in Fig. 1. 22. Becker, A., Canut, H., Luttge, U., Maeshima, M., Marigo, G.,

and Ratajczak, R. (1995) J. Plant. Physiol. 146, 88–94.23. Schwartz, M. (1971) Eur. J. Biochem. 21, 191–198.24. Avraham, Y., Grossowicz, N., and Yashphe, J. (1990) Biochim.REFERENCES

Biophys. Acta 1040, 287–293.1. Booth, J. W., and Guidotti, G. (1995) J. Biol. Chem. 270, 19377– 25. Iltzsch, M. H., el Kouni, M. H., and Cha, S. (1985) Biochemistry

19383. 24, 6799–6807.2. Brattin, W. J., and Waller, R. L. (1983) J. Biol. Chem. 258, 6724– 26. Blank, J. G., and Hoffee, P. A. (1975) Arch. Biochim. Biophys.

6729. Acta 168, 259–265.3. Banik, U., and Roy, S. (1990) Biochem. J. 266, 611–614. 27. Chanson, A., Fichmann, J., Spear, D., and Taiz, L. (1985) Plant

Physiol. 79, 159–164.4. Trentham, D. R., Bardsley, R. G., Eccleston, J. F., and Weeds,28. Rea, P. A., and Poole, R. J. (1985) Plant Physiol. 77, 46–52.A. G. (1972) Biochem. J. 126, 635–644.29. Gordon-Weeks, R., Steele, S. H., and Leigh, R. A. (1996) Plant5. Webb, M. R. (1992) Proc. Natl. Acad. Sci. USA 89, 4884–4887.

Physiol. 111, 195–202.6. De Groot, H., De Groot, H., and Noll, T. (1985) Biochem. J. 230,30. Fraichard, A., Trossat, C., Perotti, E., and Pugin, A. (1996) Bio-255–260.

chimie 78, 259–266.7. Fiske, C. H., and Subbarow, Y. (1925) J. Biol. Chem. 66, 375–

31. Vianello, A., Zancani, M., Casolo, V., and Macri, F. (1997) Plant400.Cell Physiol. 38(1), 87–90.

8. Ames, B. N. (1963) Methods Enzymol. 8, 115–118.32. Nakamura, Y., Sakakibara, Y., Kobayashi, H., and Kasamo, K.

9. Peterson, G. L. (1978) Anal. Biochem. 84, 164–172. (1997) Plant Cell Physiol. 38(3), 371–374.10. Lanzetta, P. A., Alvarez, L. J., Reinach, P. S., and Candia, O. A. 33. Wang, Y., Leigh, R. A., Kaestner, K. H., and Sze, H. (1986) Plant

(1979) Anal. Biochem. 100, 95–97. Physiol. 81, 497–502.11. Baykov, A. A., and Avaeva, S. M. (1981) Anal. Biochem. 116, 1– 34. Malsowski, P., and Maslowska, H. (1987) Biochem. Physiol.

4. Pflanzen. 182, 73–84.35. Fraichard, A., Magnin, T., Trossat, C., and Pugin, A. (1993) Plant12. Chan, K.-M., Delfert, D., and Junger, K. D. (1986) Anal. Bio-

chem. 157, 375–380. Physiol. Biochem. 31, 349–359.

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