ANALYTICAL
65, 305-309 (1975)
BIOCHEMISTRY
Rapid
Determination
Biological
of Inorganic
Systems
by a Highly
Photometric B. ANNER Departement
AND
Phosphate
in
Sensitive
Method M. MOOSMAYER
de Pharmacologic, Ecole de Ms?decine, CH-1211 Gen?ve 4, S+ritzerland
Received September 27, 1974; accepted December 24. 1974 A highly sensitive method for rapid determination of inorganic phosphate in biological tissues is described which is based on a reaction between phosphomolybdate and malachite green. Phosphate concentrations as low as l-l 6 PM can be measured, in the presence of phosphorylated compounds. Trichloracetic acid and triethanolamine can be used for extraction of phosphate from the tissue without interference in the photometric determination. The colored complex which is measured is stable for several days.
In 1947, Soyenkoff (1) published a micromethod for the determination of orthophosphate (Pi), which was based on the spectrophotometric measurement of the color change of quinaldine red in the presence of Pi, and presumably dependent on the formation of a dye-phosphomolybdate complex. Later, Altmann et al. (2) developed a similar method for the determination of Pi in water; they based their measurements on the formation of a complex between malachite green and phosphomolybdate. The present paper describes the application of this method to biological tissues. The assay is simple to carry out and allows the measurement of Pi concentrations in the range or I- 16 PM, concentrations at which Pi cannot accurately be measured by other spectrophotometric methods. The malachite green-phosphomolybdate complex was found to be very stable and Pi can be measured in solutions of various compositions. The presence of phosphorylated organic compounds does not generally interfere with the phosphate determinations. MATERIALS
The reagents were prepared molybdate (Na,MoO,. 2H,O), H,O) and sulfuric acid (Merck vinyl alcohol for synthesis Hohenbrunn, Miinchen, and
AND
according to Altmann et al. (2). Sodium sodium dihydrogenphosphate (NaH,PO . Darmstadt) were of analytical grade, polywas obtained from Merck-Schuchardt, malachite green from BDH Chemicals 305
Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
METHODS
306
ANNER
AND
MOOSMAYER
OD623nm
A
1.6 -
O0
I 4
I 8
I 12
1 16 iMPi
00623nm
0
B
8
12
16
FIG. 1. Calibration curves for inorganic phosphate determination (explanation see text). A: in water (0). B: in 5% TCA with (A) or without (A) nerve. C: in triethanolamine buffer with (m) or without (0) nerve. D: Stability of the colored complex formed at a concentration of 8 pM Pi during 5 days at 4” in water (0), TCA (a), or triethanolamine buffer (0).
Ltd, PooIe, England. Bidistilled water was used for the solutions and for rinsing the glassware. Pi standards were made in water or in the solution used for extracting inorganic phosphate from biological tissue. The optical densities were measured in a Unicam SP 1800 spectrophotometre. The procedure proposed by Altmann et al. (2) was followed except that the volume of the reaction mixture was reduced to 2.0 ml. To
314
IT0
AND
BOWMAN
angle of lo”, 20”, 30”, and 45” at 1200 and 1500 rpm at maximum flow rates of 2.5 ml/hr for 0.55 mm i.d. columns and 1.0 ml/hr for 0.3 mm i.d. columns. At lo”, all phase systems showed poor phase retention of below 30%. At 20”, the set-BuOH system gave a sufficient phase retention while other two-phase systems exhibited intensive carry-over resulting in poor phase retention. At 30” and 45”, however, all phase systems gave sufficient phase retention ranging from 30 to 50% in both column configurations. The straight helix column was found to retain a greater amount of the stationary phase at a given flow rate and revolutional speed, while the coiled helix column yielded a higher partition efficiency demonstrated by partition of bovine insulin on secBuOH/aqueous dichloroacetic acid systems. APPLICATION
TO VARIOUS
TWO-PHASE
SYSTEMS
Capability of the angle rotor (30”) has been evaluated on separations of biological materials with a variety of two-phase solvent systems of different physical properties. In order to demonstrate analytical potential of the method, a fine coiled helix column, 0.3 mm i.d. and 40 m long with a helix diameter of 1.2 mm, was used throughout unless otherwise specificed. In each separation, the column was filled with the stationary phase and a sample solution was introduced through the feed tube followed by the elution with the mobile phase using a syringe drive (Harvard Apparatus) at the indicated flow rate, while the rotor was run at the indicated revolutional speed. Separations were performed at 25°C unless otherwise indicated. Eluate was monitored through a uv monitor (LKB Uvicord II) at either 276 or 254 nm except for the polymer phase system. In separation of the colony-stimulating factor with the polymer phase system, eluted fractions were added to human bone marrow cell culture for colony count, while the total protein contents were estimated by turbidity measurement at 300 nm in 1 M perchloric acid solution. (1) Chloroform/acetic acid/O.1 N HCI (2 :2 :1) (8). This phase system has a moderately low interfacial tension, low viscosity, and a great density difference between two phases, which is ideal for countercurrent chromatography. Figure 2A shows a chromatogram of nine dinitrophenyl (DNP) amino acids at a flow rate of 0.5 ml/hr at 500 rpm, using the upper aqueous phase as a mobile phase. Sample volume was 10 ~1 containing each component in the lower phase at about 1% where solubility permits. All components were eluted out within 17 hr. Figure 2B shows a similar chromatogram obtained at a higher flow rate of 1.5 ml/hr at 1000 rpm. The separation was completed within 6 hr without serious loss of the resolution. (2) Ethyl acetate/lo% acetic acid, 5% NaCl (1 :l) (3). This phase system has a high interfacial tension and low viscosity and tends to
308
ANNER
AND
MOOSMAYER
3. Column chromatography on anion exchange resin. For more accurate measurements, the Pi was first separated from the phosphorylated organic compounds by column chromatography. In this case, the triethanolamine extraction had to be used because traces of TCA in the extract interfere with the fixation or elution of nucleotides. The extracts were layered on to small columns of Dowex-l-X8 chloride and eluted as described by Garrahan and Glynn (4). Pi was separated from ATP and adenosine 5’-diphosphate (ADP), and directly determined in the fraction eluted with 0.01 N HCl. When the Pi content of the solutions was too low, they were evaporated to dryness at 100” and taken up in a reduced volume of water. RESULTS
Standard curve in water. The optical densities found for Pi concentrations between I and 16 FM are shown in Fig. 1A. The curve shows that within these limits Pi concentrations and optical densities are linearly related. At Pi concentrations below 1 PM the measurements become difficult in view of the contamination of the blanks by ambient Pi; beyond 20 PM, the colored complex precipitates. Molar extinction coeficient. The molar extinction coefficient of the malachite green-phosphomolybdate complex in water, determined in 18 measurements, was found to amount to 99’600 (538) ODM-’ cm-’ at 623 nm, the SE is indicated in brackets. The statistical variance of the extinction coefficient is probably not due to a variability of the optical density of the colored complex, but rather to the small volumes of reagents used which somewhat impair the reproductibility of the results. The high sensitivity of the method, which amplifies Pi contaminations. may also add to the variations. Standard curves in biological extracts. In order to test whether the presence of biological extracts interferes with the Pi determination, known amounts of Pi were added to nerve extracts and the total Pi then determined. Figure 1 shows the curve obtained after substraction of the Pi found in the nerve. For Fig. lB, the TCA extract was used, for Fig. 1C the triethanolamine buffer. Both standard curves show linearity: the presence of compounds, which had been extracted from the tissue samples by TCA or in the triethanolamine buffer, appears therefore not to interfere with the Pi measurements. The measurements showed that the molar extinction coefficient of the malachite green-phosphomolybdate complex is not modified by TCA, while it is lowered by about 20% in the presence of triethanolamine. Stability of the malachite green-phosphomolybdate complex. The stability of the colored complex was measured in samples of different composition and containing different concentrations of Pi. The samples were
RAPID DETERMINATION
OF PHOSPHATE
309
stored at 4” and the optical densities checked over a period of 5 days. A typical result is shown in Fig. lD, which illustrates the high stability of the complex. DISCUSSION
Compared to the many methods for measuring Pi, the present technique offers a number of appreciable advantages. It is based on simple and relatively cheap reagents, which are commercially available and which can be used without special purification; it can be used in conjunction with solvents commonly employed for extracting tissues; it does not need separation of Pi from other compounds; it is of high sensitivity and simple and rapid to carry out; and finally, the product to be measured is so stable that reliable determinations can be made within 5 days following the initial reaction. The possible fields of application are multiple. We have used this method extensively for measuring Pi in tissue extracts (5,6), but it can also, after suitable reactions, be applied to determinations of ATPase activity, or in conjunction with selective hydrolysis and separation to measure any phosphorylated compound. ACKNOWLEDGMENTS We thank Professor R. W. Straub for help in the preparation work was supported by S.N.S.F. Grant No. 73.0890.73.
of the manuscript. The
REFERENCES 1. Soyenkoff, B. ( 1947) J. Biol. Chem. 168, 447-457. 2. Altmann. H. J.. Ftirstenau, E., Gielewski. A.. and Scholz, L. (1971) 2. Arm/. Chem. 356, 274-276.
3. Greengard, P. (1965) in Methods of Enzymatic Analysis (H. IJ. Bergmeyer, ed.). p. 552, Verlag Chemie, Weinheim, Academic Press, New York. 4. Garrahan, P. J.. and Glynn, J. M. (1967) J. Physiol. London 192, 237-256. 5. Straub. R. W., Anner, B.. Ferrero, J., and Jirouek, P. in Comparative Physiology (L. Bolis. K. Schmidt-Nielsen, and S. H. P. Maddrell, ed.), North Holland, Amsterdam. London. in press. 6. Anner. B.. Ferrero, J., Jirounek, P.. and Straub, R. W. J. Physiol. London. in press.
65, 305-309 (1975)
BIOCHEMISTRY
Rapid
Determination
Biological
of Inorganic
Systems
by a Highly
Photometric B. ANNER Departement
AND
Phosphate
in
Sensitive
Method M. MOOSMAYER
de Pharmacologic, Ecole de Ms?decine, CH-1211 Gen?ve 4, S+ritzerland
Received September 27, 1974; accepted December 24. 1974 A highly sensitive method for rapid determination of inorganic phosphate in biological tissues is described which is based on a reaction between phosphomolybdate and malachite green. Phosphate concentrations as low as l-l 6 PM can be measured, in the presence of phosphorylated compounds. Trichloracetic acid and triethanolamine can be used for extraction of phosphate from the tissue without interference in the photometric determination. The colored complex which is measured is stable for several days.
In 1947, Soyenkoff (1) published a micromethod for the determination of orthophosphate (Pi), which was based on the spectrophotometric measurement of the color change of quinaldine red in the presence of Pi, and presumably dependent on the formation of a dye-phosphomolybdate complex. Later, Altmann et al. (2) developed a similar method for the determination of Pi in water; they based their measurements on the formation of a complex between malachite green and phosphomolybdate. The present paper describes the application of this method to biological tissues. The assay is simple to carry out and allows the measurement of Pi concentrations in the range or I- 16 PM, concentrations at which Pi cannot accurately be measured by other spectrophotometric methods. The malachite green-phosphomolybdate complex was found to be very stable and Pi can be measured in solutions of various compositions. The presence of phosphorylated organic compounds does not generally interfere with the phosphate determinations. MATERIALS
The reagents were prepared molybdate (Na,MoO,. 2H,O), H,O) and sulfuric acid (Merck vinyl alcohol for synthesis Hohenbrunn, Miinchen, and
AND
according to Altmann et al. (2). Sodium sodium dihydrogenphosphate (NaH,PO . Darmstadt) were of analytical grade, polywas obtained from Merck-Schuchardt, malachite green from BDH Chemicals 305
Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
METHODS
306
ANNER
AND
MOOSMAYER
OD623nm
A
1.6 -
O0
I 4
I 8
I 12
1 16 iMPi
00623nm
0
B
8
12
16
FIG. 1. Calibration curves for inorganic phosphate determination (explanation see text). A: in water (0). B: in 5% TCA with (A) or without (A) nerve. C: in triethanolamine buffer with (m) or without (0) nerve. D: Stability of the colored complex formed at a concentration of 8 pM Pi during 5 days at 4” in water (0), TCA (a), or triethanolamine buffer (0).
Ltd, PooIe, England. Bidistilled water was used for the solutions and for rinsing the glassware. Pi standards were made in water or in the solution used for extracting inorganic phosphate from biological tissue. The optical densities were measured in a Unicam SP 1800 spectrophotometre. The procedure proposed by Altmann et al. (2) was followed except that the volume of the reaction mixture was reduced to 2.0 ml. To
314
IT0
AND
BOWMAN
angle of lo”, 20”, 30”, and 45” at 1200 and 1500 rpm at maximum flow rates of 2.5 ml/hr for 0.55 mm i.d. columns and 1.0 ml/hr for 0.3 mm i.d. columns. At lo”, all phase systems showed poor phase retention of below 30%. At 20”, the set-BuOH system gave a sufficient phase retention while other two-phase systems exhibited intensive carry-over resulting in poor phase retention. At 30” and 45”, however, all phase systems gave sufficient phase retention ranging from 30 to 50% in both column configurations. The straight helix column was found to retain a greater amount of the stationary phase at a given flow rate and revolutional speed, while the coiled helix column yielded a higher partition efficiency demonstrated by partition of bovine insulin on secBuOH/aqueous dichloroacetic acid systems. APPLICATION
TO VARIOUS
TWO-PHASE
SYSTEMS
Capability of the angle rotor (30”) has been evaluated on separations of biological materials with a variety of two-phase solvent systems of different physical properties. In order to demonstrate analytical potential of the method, a fine coiled helix column, 0.3 mm i.d. and 40 m long with a helix diameter of 1.2 mm, was used throughout unless otherwise specificed. In each separation, the column was filled with the stationary phase and a sample solution was introduced through the feed tube followed by the elution with the mobile phase using a syringe drive (Harvard Apparatus) at the indicated flow rate, while the rotor was run at the indicated revolutional speed. Separations were performed at 25°C unless otherwise indicated. Eluate was monitored through a uv monitor (LKB Uvicord II) at either 276 or 254 nm except for the polymer phase system. In separation of the colony-stimulating factor with the polymer phase system, eluted fractions were added to human bone marrow cell culture for colony count, while the total protein contents were estimated by turbidity measurement at 300 nm in 1 M perchloric acid solution. (1) Chloroform/acetic acid/O.1 N HCI (2 :2 :1) (8). This phase system has a moderately low interfacial tension, low viscosity, and a great density difference between two phases, which is ideal for countercurrent chromatography. Figure 2A shows a chromatogram of nine dinitrophenyl (DNP) amino acids at a flow rate of 0.5 ml/hr at 500 rpm, using the upper aqueous phase as a mobile phase. Sample volume was 10 ~1 containing each component in the lower phase at about 1% where solubility permits. All components were eluted out within 17 hr. Figure 2B shows a similar chromatogram obtained at a higher flow rate of 1.5 ml/hr at 1000 rpm. The separation was completed within 6 hr without serious loss of the resolution. (2) Ethyl acetate/lo% acetic acid, 5% NaCl (1 :l) (3). This phase system has a high interfacial tension and low viscosity and tends to
308
ANNER
AND
MOOSMAYER
3. Column chromatography on anion exchange resin. For more accurate measurements, the Pi was first separated from the phosphorylated organic compounds by column chromatography. In this case, the triethanolamine extraction had to be used because traces of TCA in the extract interfere with the fixation or elution of nucleotides. The extracts were layered on to small columns of Dowex-l-X8 chloride and eluted as described by Garrahan and Glynn (4). Pi was separated from ATP and adenosine 5’-diphosphate (ADP), and directly determined in the fraction eluted with 0.01 N HCl. When the Pi content of the solutions was too low, they were evaporated to dryness at 100” and taken up in a reduced volume of water. RESULTS
Standard curve in water. The optical densities found for Pi concentrations between I and 16 FM are shown in Fig. 1A. The curve shows that within these limits Pi concentrations and optical densities are linearly related. At Pi concentrations below 1 PM the measurements become difficult in view of the contamination of the blanks by ambient Pi; beyond 20 PM, the colored complex precipitates. Molar extinction coeficient. The molar extinction coefficient of the malachite green-phosphomolybdate complex in water, determined in 18 measurements, was found to amount to 99’600 (538) ODM-’ cm-’ at 623 nm, the SE is indicated in brackets. The statistical variance of the extinction coefficient is probably not due to a variability of the optical density of the colored complex, but rather to the small volumes of reagents used which somewhat impair the reproductibility of the results. The high sensitivity of the method, which amplifies Pi contaminations. may also add to the variations. Standard curves in biological extracts. In order to test whether the presence of biological extracts interferes with the Pi determination, known amounts of Pi were added to nerve extracts and the total Pi then determined. Figure 1 shows the curve obtained after substraction of the Pi found in the nerve. For Fig. lB, the TCA extract was used, for Fig. 1C the triethanolamine buffer. Both standard curves show linearity: the presence of compounds, which had been extracted from the tissue samples by TCA or in the triethanolamine buffer, appears therefore not to interfere with the Pi measurements. The measurements showed that the molar extinction coefficient of the malachite green-phosphomolybdate complex is not modified by TCA, while it is lowered by about 20% in the presence of triethanolamine. Stability of the malachite green-phosphomolybdate complex. The stability of the colored complex was measured in samples of different composition and containing different concentrations of Pi. The samples were
RAPID DETERMINATION
OF PHOSPHATE
309
stored at 4” and the optical densities checked over a period of 5 days. A typical result is shown in Fig. lD, which illustrates the high stability of the complex. DISCUSSION
Compared to the many methods for measuring Pi, the present technique offers a number of appreciable advantages. It is based on simple and relatively cheap reagents, which are commercially available and which can be used without special purification; it can be used in conjunction with solvents commonly employed for extracting tissues; it does not need separation of Pi from other compounds; it is of high sensitivity and simple and rapid to carry out; and finally, the product to be measured is so stable that reliable determinations can be made within 5 days following the initial reaction. The possible fields of application are multiple. We have used this method extensively for measuring Pi in tissue extracts (5,6), but it can also, after suitable reactions, be applied to determinations of ATPase activity, or in conjunction with selective hydrolysis and separation to measure any phosphorylated compound. ACKNOWLEDGMENTS We thank Professor R. W. Straub for help in the preparation work was supported by S.N.S.F. Grant No. 73.0890.73.
of the manuscript. The
REFERENCES 1. Soyenkoff, B. ( 1947) J. Biol. Chem. 168, 447-457. 2. Altmann. H. J.. Ftirstenau, E., Gielewski. A.. and Scholz, L. (1971) 2. Arm/. Chem. 356, 274-276.
3. Greengard, P. (1965) in Methods of Enzymatic Analysis (H. IJ. Bergmeyer, ed.). p. 552, Verlag Chemie, Weinheim, Academic Press, New York. 4. Garrahan, P. J.. and Glynn, J. M. (1967) J. Physiol. London 192, 237-256. 5. Straub. R. W., Anner, B.. Ferrero, J., and Jirouek, P. in Comparative Physiology (L. Bolis. K. Schmidt-Nielsen, and S. H. P. Maddrell, ed.), North Holland, Amsterdam. London. in press. 6. Anner. B.. Ferrero, J., Jirounek, P.. and Straub, R. W. J. Physiol. London. in press.
