ANALYTICAL

BIOCHEMISTRY

84,

164-172 (1978)

A Simplified Method for Analysis of inorganic Phosphate in the Presence of interfering Substances
GARY L. PETERSON Department of Pharmacology, University of Wisconsift, Ma&m, Wisconsin 53706

Received June 20, 1977; accepted September 2, 1977 A new phosphate analysis method is described which is designed primarily for use with routine phosphatase assays. The method is modified extensively from that of Fiske and SubbaRow [(1925) J. Bid. C&em, 66, 3753 by decreasing 10fold the level of reducing agent, ANSA, increasing the concen~ation of ammonium molybdate to 4 mM, utilizing HCI as the only acid present, and incorporating SDS. The method is insensitive to pH, ATP, proteins, and detergents at concentrations normally encountered in enzyme purification and analysis experiments. The color developed is fivefold more stable than that from the originat Fiske-SubbaRow method, and the standard curve retains linearity for at least 24 hr with acid concentrations between 0.6 and 1.0 N HCI and for waveIen@hs between 500 and 900 nm. The simplicity, stability, and general applicability of the method allow for convenient and rapid analysis of large numbers of samples under a wide variety of conditions.

Measurement of inorganic phosphate is the basis of many enzyme assays. Due to its simplicity, one of the most popular spectrophotometric methods for the determination of inorganic phosphate is that of Fiske and SubbaRow (I). However, certain enzyme assay conditions interfere with this method as well as other spectrophotometric methods [see reviews in Refs. (2,3)j. For instance, buffers used in enzyme assays can interfere with phosphate analysis by altering the pH (4). Substances including ATP, ADP, AMP, FPit glycerol phosphate (5-g), organic acids (4,9,10), mannitol, and sorbitol (11) can interfere by competing for the free molybdate. Interference by these substances can be reduced by increasing the concentration of ammonium molybdate in the phosphate assay above 3 mM (4-11). Proteins (12,13), amine drugs (14,15), and low concentrations of detergents (12-15) often produce turbidity in the final color solution in the phosphate assay. Proteins are usually removed prior to phosphate analysis by acid precipitation (14). Amine and detergent interference can be removed by silicotungstie-perchloric acid precipitation (41,
Abbreviations used: ATP, ADP, and AMP, adenosine tri-, di-, and monophosphate; PP,, inorganic pyrophosphate; ANSA, I-amino-Z-naphthol-4-sulfonic acid; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid. ooO3-2697178/084f-0164$02.00/O
Copyr?ght 0 1978 by Academic Press, Inc. All rights of reproduction in any fotm reserved.

164

A SIMPLIFIED

PHOSPHATE

ASSAY

165

by chloroform extraction, or by addition of alcohol (15). More recently, high concentrations of SDS were found to prevent the turbidity caused by proteins and detergents (12,13,16). An additional problem peculiar to the Fiske-SubbaRow method is that the color continues to increase with time. Huxtable and Bressler (5) have shown that the final color reaction in their modified Fiske-SubbaRow method is stabilized by addition of an arsenite-citrate solution. Unfortunately, none of the modified methods treat all of the common types of problems discussed above. This study was undertaken in an effort to arrive at a single method for alleviating these problems while retaining the simplicity of the original Fiske-SubbaRow method. Preliminary experiments showed that the Huxtable and Bressler (5) method was unaffected by the addition of SDS (12,13), but was highly sensitive to pH, particularly when the molybdate concentration was increased. Subsequent studies indicated that satisfactory color stability could be achieved by lowering the concentration of the reducing agent, ANSA, in a modified Fiske-SubbaRow method containing 4 mM ammonium molybdate and 1.8% SDS. For convenience, HCl was substituted for H,SO, and TCA to produce the acidic conditions in the modified method. This paper describes this new modified phosphate analysis method and the experiments designed to determine the optimum phosphate assay conditions. The new method is simple, rapid, relatively stable with time, and substantially free of the types of interferences described above.
MATERIALS AND METHODS

Materials. ANSA from Sigma was recrystallized and prepared fresh monthly as a 0.25% (w/v) stock solution, as described by Fiske and SubbaRow (l), and stored in the dark at room temperature. SDS and BSA were obtained from Sigma and used as received. Na,ATP was obtained from PL Biochemicals, Inc. Membrane proteins were prepared from the nauplius larvae of the brine shrimp by isolation of a particulate fraction obtained by centrifugation of a 5OOOg, lo-min supernatant fraction at 48,OOOg for 30 min. The resulting pellet was washed twice with distilled water by homogenization and centrifugation at 48,000g for 30 min. Protein concentration of the membrane fraction was estimated by a modification (17) of the method of Lowry et al. (18) using crystalline BSA as the standard. All other reagents were analytical reagent grade. Phosphate analysis method. For a final assay volume of 5.0 ml, bring the sample to 3.0 ml with distilled water, then add 1.0 ml of acid molybdate solution (2.5% ammonium molybdate in 4 N HCl), 0.8 ml of 10% SDS, and 0.2 ml of 0.025% ANSA (0.25% stock ANSA diluted l/10 in HzO, prepared fresh weekly). Mix and allow to stand at about 20C for at least 30 min before reading absorbance at 700 nm. Subsequent

166

GARY

L. PETERSON

color development is less than 1% per hour. The sequence of addition of acid molybdate and SDS may be reversed, and volumes and concentrations of stock reagents may be altered as convenient. For example, for 0.5ml enzyme reactions, I use 0.9 ml of 5% SDS, 1.0 ml of 1.25% ammonium molybdate in 2 N HCl, and 0.1 ml of 0.025% ANSA. Optimum conditions for the phosphate assay were determined by examination of the effects of pH, ANSA, SDS, and time on extinction, linearity of the standard curve, and the absorption spectrum. Details of these experiments are indicated in the figures and figure legends. All the indicated concentrations are those in the final assay mixture. This convention is adhered to throughout the remainder of the manuscript unless otherwise specified. The new phosphate analysis method was compared to the method of Fiske and SubbaRow (1) with respect to extinction and color stability, and to the methods of Fiske and SubbaRow and Huxtable and Bressler (5) with respect to the effects of interfering substances. Two variations using either 0.5 N H,SO, or 4% TCA plus 0.3 N HzS04, as reported by Fiske and SubbaRow, were tested. Final phosphate assay volumes were 5.0 ml for the new method and the Fiske-SubbaRow method, and 4.7 ml as described for the Huxtable-Bressler method.
RESULTS AND DISCUSSION

The new phosphate assay method is relatively insensitive to changes in HCl concentration from 0.6 to 1.2 N (Fig. 1). Within this range of HCl concentration, the concentration of the reducing agent, ANSA, has little effect on the initial extinction for phosphate. However, the timedependent change in the phosphate extinction decreases markedly at lower concentrations of ANSA. At the same time, the lower ANSA concentrations result in some pH sensitivity, as evidenced by a greater time-dependent change in phosphate extinction at the higher concentrations of HCl. The compromise concentrations of HCI and ANSA chosen (0.8 N and 1.O mg%, respectively) give a minimum color change with time, together with a less than 2.5% maximum error in extinction, with samples between 0.6 and 1.0 N HCl. Furthermore, this range of pH is compatible with minimum nonenzymatic hydrolysis of several phosphate substrates (including ATP) in the presence of ammonium molybdate (19). The small differences in the pH and time curves for 1.25 and 0.5 mg% ANSA (Fig. 1) demonstrate that the absolute amount of ANSA at these low concentrations is not critical. The presence of SDS does not effect the phosphate assay in terms of extinction, color stability, or linearity of the standard curve (Fig. 2). These findings are in agreement with other reports (12,13,16). Figure 2 (insets) also demonstrates that HCl concentrations between 0.6 and 1.O N

A SIMPLIFIED

PHOSPHATE

ASSAY

167

0.4

0.6 HCI

1.2

1.6

0.4

0.6 (NJ

1.2

0.5 h

1.6

Concentration

FIG. 1. The effect of pH and ANSA concentration on phosphate extinction (absorbance at 725 nm for 80 PM NaH,FQ,) at different time points after completion of the assay. The time in hours is shown by the numerals to the right of each curve. All samples are controlled with appropriate reagent blanks.

do not effect the standard curve for at least 6 hr, with or without the presence of SDS. Further, the reagent blanks show little color development over a 6-hr period, regardless of the concentration of HCl or the presence of SDS. The absorption spectrum shifts from an initial maximum near 750 nm to a prominent peak near 820 nm (Fig. 3). The data in Fig. 3 were obtained in the presence of 1.0 N HCl, where color stability with time is reduced. Nevertheless, the standard curve for phosphate remains linear between 660 and 820 nm (Fig. 3, inset) as well as other wavelengths (data not shown). These findings are in contrast with observations made with the Fiske-SubbaRow method, where there was a loss of linearity in the standard curve by 3 hr, especially at longer wavelengths. The extinction for phosphate by the new method is somewhat lower than that for the original Fiske-SubbaRow method, but is fivefold more stable (Table 1). The variation of the Fiske-SubbaRow method which contains only 0.5 N H,SO, appears more stable than the variation containing 4% TCA plus 0.3 N H,SO,. With both variants, however, the standard curve became nonlinear with time, particularily at the longer

168
0.6N *$JJ

GARY L. PETERSON
HCI m d:;=J l.ON HCI m

+z 0.0E o6

0.2 0.4 0
Phoaphatr

0.2 0.4
(umoler) ytso

0.2 0.4 Phoaphata

0.2 (rmolra)

0.4

/++----60

Tpee,20
,f-----60

ir~-Aw3
0 ,l------60 -,*O

g 0.4: n k a 2

o.2;p~e40

4o

Time

(hours)

FIG. 2. The effect of the presence (open circles) and absence (closed circles) of 1.8% SDS on absorbance at 750 nm (A,& for 0, 40, 80, 120, and 160 pM phosphate between 30 min and 6 hr of incubation in the presence of 0.6 and 1.0 N HCI. The insets at the top show the standard curves for phosphate at the 30-min and 6-hr time points for a total assay volume of 2.5 ml. The absorbance of the reagent blank was read against H20, whereas the phosphate standards were read against their appropriate reagent blanks. wavelengths, and a precipitate appeared by 2 hr in the system containing only 0.5 N H,SO,. The data in Table 1 demonstrate that the maximum extinction compatible with the greatest stability of the final color reaction by the new method occurs at 700 to 750 nm. At 750 nm, the extinction is slightly higher and the spectrophotometer slit width narrower, but the rate of color change is about 2% per hour as opposed to about 1% per hour at 700 nm. At either wavelength, samples read within 1 hr of the standards will be within experimental error. The effects of various interfering substances on the analysis of phosphate by the new method and the methods of Fiske and SubbaRow (1) and Huxtable and Bressler (5) are compared in Table 2. The two variations of Fiske and SubbaRow are equivalent to the new method in tolerating added HCl and BSA, but inferior with respect to the other substances tested. The Huxtable and Bressler method is superior in its tolerance to ATP, but undesirable in the presence of other interfering substances. The new method, however, is substantially insensitive to all of these interfering substances. Levels of acid or base changes, ATP, proteins, or detergents expected from enzyme studies can be tolerated without time-consuming extraction or centrifugation steps. Thus, because of its simplicity, general applicability, and stability, the new method is very

A SIMPLIFIED

PHOSPHATE

ASSAY

169

0.7 t

PhOSphO+e

(rmoles)

Wavelength

(nm)

FIG. 3. Absorption spectra for 80 pM phosphate after 1,6, and 21 hr of incubation at 20C. The insets above show the standard curves for phosphate at 660, 700, 750, and 820 nm after 1 hr (closed circles) and 21 hr (open circles) of incubation. The concentration of HCl was 1.0 N, and the total volume was 5.0 ml.

suitable for the rapid analysis of large numbers of samples, under a wide variety of conditions. The presence of ATP, or similar phosphatase substrates in the assay for phosphate, requires additional considerations. ATP competition for the free molybdic acid has been minimized in the new assay method by raising the ammonium molybdate concentration to about 4 mM (6-8). This type of ATP interference remains sensitive to pH. Color development in the presence of 1.0 N HCl is near 95 and 80% of control levels at 1 and 2 mM ATP, respectively, whereas in the presence of 0.6 N HCI it is still above 90% at 4 mM ATP. ATP interference is considered tolerable at 2 mM in the presence of 0.8 N HCl. The absolute amount of ATP in a sample can be increased by increasing the total volume of the assay, although a loss of sensitivity would occur.

170

GARY L. PETERSON TABLE 1

WITH

COMPARISON THAT

OF THE EXTINCTION AND OF FISKE AND SUBBAROW

COLOR STABILITY OF THE NEW METHOD (1) AT FOUR DIFFERENT WAVELENGTHS

Fiske and SubbaRow Wavelength 0.5 N H,SO, E: ,m,M(%Alhr)b 4.02 4.45 4.82 5.40 (3.23) (3.37) (7.67) (15.5) 4% TCA + 0.3 E: ,,M(%&hr) 3.98 4.40 4.82 5.28
N

New method H$O, 0.6 N HCI E: F$(%A/hr) 3.55 3.93 4.03 3.94 (0.92) (0.73) (1.50) (2.87) 1.0 N HCI E: ?$(%A/hr) 3.48 3.80 3.94 3.56 (0.82) (1.28) (2.02) (2.68)

(nm)
660 700 750 820

(5.91) (6.14) (8.91) (17.2)

a Phosphate was analyzed by two variations suggested by Fiske and SubbaRow which differ in the acid additions (see Methods for more details). b The millimolar extinction (cmVmmo1) is calculated after 1 hr of color development and the percentage change in extinction per hour is calculated between 1 and 6 hr ofcolor development at 16C.

Perchloric acid (8) or TCA (20) used for protein precipitation aggravates the ATP interference by reducing the pH. However, with the new method, protein removal is usually not necessary, as up to 200 pg in a 5-ml assay volume can be tolerated. In preparations where this level is exceeded, such as in phosphatase assays of low activity, the protein or a portion of it must be removed. If the amount of protein is only slightly excessive, sufficient protein may be removed by precipitation with HCl, which in this modification is prepared and added separately from the ammonium molybdate. With extremely excessive amounts of protein, adequate precipitation is achieved by addition of 0.10 ml of 50% TCA/l .O ml of sample, the only effect being to reduce the ATP tolerance to 1 mM. The additional 0. lo-ml volume is compensated within experimental error by the volume of the resulting pellet. ATP interference has also been reported to be aggravated by potassium (6). With the new method, a pH-dependent K interference was observed. A precipitate forms at K concentrations greater than 75 and 160 mM for 0.6 and 1 .O N HCl, respectively. However, ATP and K together were found to slightly reduce their individual effects on the assay. ATP further interferes with the phosphate assay by undergoing hydrolysis in the presence of the acid molybdate. The phosphate produced by hydrolysis contributed to continued color development, which by the new method is linear for at least 6 hr at 20C and dependent on pH (0.10 and 0.15 A,, units/hr with 1 mM ATP for 0.6 and 1.0 N HCI, respectively). However, the differential amount of color between blanks and standard amounts of phosphate remains the same in the presence or absence of ATP. Thus, it is only important that the appropriate controls be read at

A SIMPLIFIED

PHOSPHATE TABLE 2

ASSAY

17I

COMPARISON BY THE

OF THE NEW

EFFECTS

OF INTERFERING

SUBSTANCES

ON PHOSPHATE SUBBAROW

ANALYSIS (1)

METHOD AND THE AND HUXTABLE

METHODS OF FISKE AND AND BRESSLER (5)

Percentage of control absorbance (750 nm)a Fiske-SubbaRow Added substance None

HCl

Final concentration Control 0.2 N 0.2 N

1 rnM

&SO, 100.0 103.5 90.3 89.0 84.5 102.8 102.8

loo.Y

TCA-H,SO, 100.0 101.0 92.3 92.2 88.5 104.6 106.0 100.1 91.4 90.6 7.4 59.0 4.7 90.1

HuxtableBressler 100.0 134.0 0.8 103.9 98.4 107.8 93.7 103.7 100.5 154.5 360.4 185.5 378.2 95.0

New method
100.0

NaOH ATP BSA Membrane protein Lubrol WX Triton X-100 NaK-ATPase mix

2rnM

0.02 mg/ml 0.04 mg/ml 0.02 mg/ml 0.04 mg/ml 0.20 mgiml 0.50 mg/ml 0.08 mgiml 0.80 mg/ml (1 ml)

85.y 76.5 175.7 30.5

0.0

87.3

101.7 97.1 95.1 93.6 102.6 106.8 100.0 105.1 98.6 103.8 100.0 98.5 95.0

D Samples containing 40 PM phosphate were prepared in duplicate and read against their appropriate reagent blanks. * Two variations of the Fiske-SubbaRow method (see Methods for more details). c Samples centrifuged to remove particulate material before reading absorbance. I NaK-ATPase mix consists of l.O-ml sample of 5 mM Nlt,ATP, 10 mM MgCl,, 160 mM NaCl, 40 mMKC1, 100 mM imidazole-HCl buffer, pH 7.2.

the same time as the phosphate samples, and that the samples be read before too much color from ATP hydrolysis accumulates (2 to 3 hr at room temperature). If ATP is present and it is inconvenient to complete the phosphate analysis within 2 to 3 hr, the samples may be stored in either of two ways: (i) The enzyme assay can be terminated by the addition of SDS, and the samples stored at this point where ATP hydrolysis would be minimal. (ii) The phosphate assay solutions can be added as usual and the samples stored at 0C until absorbances can be read. ATP hydrolysis then contributes less than 0.03 AT5,, units/hr with 1 mM ATP, which is tolerable for overnight storage. Before reading the absorbance, the samples must be warmed to room temperature to dissolve the cold-precipitated SDS and the 30-min color development time must be allowed for if previously omitted. In either event, these considerations circumvent the complication of extracting the phosphomolybdic acid complex to avoid ATP hydrolysis (2,3).

172

GARY L. PETERSON

ACKNOWLEDGMENTS
I wish to express my gratitude to Drs. Lowell E. Hokin, Lynn Churchill, and Eugene Quist for their helpful suggestions during preparation of this manuscript. This work was supported by grants from the National Institutes of Health (HL-16218) and the National Science Foundation (GB-40368X) to Dr. Lowell E. Hokin.

REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Fiske, C. H., and SubbaRow, Y. (1925) J. Viol. Chem. 66, 375-400. Lindberg, O., and Ernster, L. (1956) Methods Biochem. Anal. 3, l-22. Sanui, H. (1974) Anal. Biochem. 60, 489-504. Gomori, G. (1941-1942) J. Lab. C/in. Med. 27, 955-960. Huxtable, R., and Bressler, R. (1973) Anal. Biochem. 54, 604-608. Blum, J. J., and Chambers, K. W. (1955) Biochim. Biophys. Acra 18, 601. Kushmerick, M. J. (1972) Anal. Biochem. 46, 129-134. Seddon, B., and Fynn, G. H. (1973) Anal. Biochem. 56, 566-570. Berenblum, I., and Chain, E. (1938) Biochem. J. 32, 286-294. Davies, D. R., and Davies, W. C. (1932) Biochem. J. 26, 2046-2055. Ho, C. H., and Pande, S. V. (1974) Anal. Biochem. 60, 413-416. Dulley, J. R. (1975) Anal. Biochem. 67, 91-96. Tashima, Y. (1975) Anal. Biochem. 69, 410-414. Roufogalis, B. D. (1971) Anal. Biochem. 44, 325-328. Ueda, I., and Wada, T. (1970) Anal. Biochem. 37, 169-174. Atkinson, A., Gatenby, A. D., and Lowe, A. G. (1973) Biochim. Biophys. Acta 320, 195-204. Peterson, G. L. (1977) Anal. Biochem., in press. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. Weil-Malherbe, H., and Green, R. H. (1951) Biochem. J. 49, 286-292. Hwang, K. J. (1976) Anal. Biochem. 75, 40-44.