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Orthophosphate analysis by the Fiske-SubbaRow method and interference by adenosine phosphates and pyrophosphate at variable acid pH
ANALYTICAL
56, 566-570 (1973)
BIOCHEMISTRY
Orthophosphate Analysis by the Fiske-SubbaRow and Interference by Adenosine Phosphates Pyrophosphate
at Variable
B. SEDDON’ institute
Department of Science
of
Biochemistry,
and Technology,
Received
March
AND
Acid
Method and
pH
G. H. FYNN
University Manchester
of Manchester, M6’0 l&D, United
2, 1973; accepted
Kingdom
June 28, 1973
ATP, ADP, AMP, and pyrophosphate interfere with colour development in the Fiske-SubbaRow assay procedure. The extent of interference observed is dependent on the pH of the final assay system; small changes in pH at low pH values lead to large variations in the amount of ATP interference. Addition of excess ammonium molybdate and neutralisation of any previous acid additions (such that the pH is maintained constant throughout all assay procedures) removes interference and Pi can be estimated in the presence of relatively high ATP concentrations. In previous reports containing compounds
(14) form
it has been noted that various phosphatecomplexes with molybdic acid. In a recent
communication (1) Kushmerick has reported upon the interference with Pi analysis by ATP, AMP, pyrophosphate, and phosphorylcreatine when using the method of Berenblum and Chain (5) and the removal of such interferences by adding extra molyb’date. Previously Blum and Chambers (‘2) had reported on the interference by ATP, ADP, and ITP in the FiskeSubbaRow analysis and the removal of such interference by addition of extra molybdate. The interference was due to the formation of a colourless complex between ATP and molybdate which did not become coloured upon the addition of reducing agent and catalyst. During investigations of bacterial ATPase systems we have also observed interference with Pi analysis by ATP, ADP, and AMP, and pyrophosphate using the Fiske-SubbaRow assay procedure. While the removal of such interference by addition of extra molybdate has already been reported (1,2) we noticed that, with the Fiske-Subb8aRow method, such factors as pH and the type and phosphate content of the phosphatecontaining
compound
influenced
the extent
’ Present address: Department of Developmental Marischal College, Aberdeen, AB9 lAS, U.K. 566 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
of interference
obtained
Biology, University
with
of Aberdeen,
ORTHOPHOSPHATE
ANALYSIS
567
this assay system. Conditions (which are reported here) were adopted such that assay of ATPase activity by measuring the release of Pi could be monitored even with concent,rat,ions of ATP which interfered with the original Fiske-SubbaRow assay system. METHODS
Inorganic orthophosphate (Pi) was measured by the Fiske-SubbaRow procedure (6). The assay system contained O-4.4 ml sample; 0.4 ml 20 mM (or 40 mM, see Result’s section) ammonium molybdate in 5 P; H&30, ; 0.2 ml 1-amino-2-napthol-4-sulphonic acid ( ANS) in bisulphite solution (6)) and distilled water to a total volume of 5.0 ml. The colour was estimated at 660 nm after 17-min development using a Unicam SP 600 spectrophotometer with a lightpath of 1 cm. In experiments in which the effect of ATP was studied ATP (disodium salt,, Sigma Chemical Co.) at the concentration given in the Results section, was also added. The effects of ADP, AMP, (Boehringer Co. Ltd.), pyrophosphate, and adenosine were investigated by replacing ATP with the above components. During the investigations of bacterial ATPase activity enzyme-catalysed reactions were stopped by the addition of an equal volume of HClO, (0.7 M). In order t’o mimic these conditions Pi assays in t,he presence and absence of ATP were performed with HClO, added. Assays were also made on test systems where the added HClO, was neut,ralised by addition of NaOH. In all assay procedures tubes were allowed to stand for 10 min at’ 0°C after addition of HClO, (release of Pi by acid hydrolysis of ATP by this treatment was tested for by including incubations with ATP alone). Pi content was then estimated by the method of Fiske-SubbaRow (6) as described above. All values quoted in the results section refer to the concentration of components in the final 5.0-ml assay system unless stated otherwise. RESULTS
AND
DISCUSSION
Initial studies estimating Pi content in the presence of ATP gave variable values for Pi content. It soon became apparent that this was a result of pH variance since the lowest values obtained for Pi in the presence of a fixed amount of ATP were those obtained using largervolume aliquots (approaching 4.4 ml) of HClO,-treated samples. Higher values for Pi were obtained with low-volume aliquots of HClO,-t,reated samples and the highest values of all were obtained when HClO, had subsequently been neutralised with NaOH. Figure 1 shows the estimation of a fixed amount of Pi with different, ATP content (i) in the absence of HClO,, (ii) in the presence of HClO, (0.28 M), and (iii) in the
SEDDON AND FYNN
568
00
L
,, 4
AT&I)
FIG. 1. The effect of ATP concentratron and acid content on colour development in the Fiske-SubbaRow estimation of inorganic phosphate. The standard assay system contained Pi, 0.5 amoles and was assayed by the Fiske-SubbaRow procedure as described in the Methods section. ATP concentration was varied as shown in the figure. Assay procedure; 0, in the p r esence of HClO, (0.28 M) ; 0, in the absence of HCIOa; A, added HClO, neutralised with NaOH before assay; ---, assay in the absence of ATP with HClO, (0.28 M) added.
presence of HClO, neutralised with NaOH. It can be seen that interference of Pi determination increased with increasing concentration of ATP and that this interference was more pronounced in the presence of HClO,. Additions of HClO, concentrations up to 0.31 M did not interfere with the Pi assay test system when ATP was absent. The presence of HClO, resulted in a lowering of pH of the test system (pH of test system approximately 1.0-1.3). It is this decrease in pH which increases the interference of Pi assay brought about by ATP since neutralisation of HClO, reduced the interference of ATP to values similar to those observed in the absence of HCIO, (Fig. 11. The amount of ammonium molybdate present in the final test system is approximately 8 pmoles. At high and low pH values 16 pmoles and 12 pmoles ATP, respectively, completely abolished any “molybdenum blue” formation (Fig. 1). Thus, ammonium molybdate:ATP ratios 1:2 and 1: 1.5 effectively removed free ammonium molybdate from the test systems. Alberty, Smith, and Bock (71 have discussed the pk values of the phosphate groups of ATP and it is noticeable from their work that the primary phosphate groups have a pk value <2.0. Changes in pH therefore, at values <2.0 would influence the balance of ionised to nonionised
primary
phosphate
groups
and
thus
such groups could interfere with the assay of Pi.
the
extent
to
which
ORTHOPHOSPHATE
The
Additions
Effects
of Various
to assay system
Compounds
TABLE 1 on the Fiske-SubbaRow
of 0.5 pmoles
Concn
of additive 5.0 ml
Pi
ATP ATP ATP + HClOd ATP + ammonium ADP AMP Pyrophosphate Adenosine a The Fiske-SubbaRow using 20 mM ammonium in the table above.
569
ANALYSIS
1
Assay
in final
of Pi” y0 Inhibition of colour development at 660 nm
3rnM
0 100 20 0 50
3rnM 3rnM
12 100
4rnM
0
rnM
3rnM
molybdate
assay procedure molybdate (final
1 mM + 0.28 rnM 3rnM$3.2rnM
was as described in the Methods concentration 1.6 mM) except where
se&ion stated
Further studies with ADP, AMP, pyrophosphate, and adenosine (Table 1) indicated that the presence of an ionisable phosphate group was, in fact, responsible for the interference observed. The greater the number of ionisable phosphate groups present the stronger was the interference observed. Aden,osine did not show any interfering properties. It is assumed, as has been reported previously (1,2), that the various phosphate-containing compounds are reacting with t-he ammonium molybdate through their ionisable phosphate groups to form colourless complexes, thus rendering the ammonium molybdate inaccessible for reaction with Pi. Table 1 shows that increasing the final concentration of ammonium molybdate from 1.6 rnM to 3.2 rnM completely removed the interference due to ATP. Increasing the final concentration of ANS reagent did not restore “molybdenum blue” formation. As a result of the above findings and the possibility that samples for Pi assay may contain high concentrations of ATP, 3.2 mM final concentration of ammonium molybdate was used in the assay system for Pi estimation. Figure 2 shows the calibration curves for assays containing (i) 1.6 rnM ammonium molybdate (ii) 3.2 rnM ammonium molybdate and (iii) 3.2 mM ammonium molybdate in the presence of 10 @moles ATP. The modification employed did not alter the sensitivity nor the characteristics of the calibration curve. Increasing the concentration of ammonium molybdate such that the ammonium molybdate: ATP ratio is 5 2: 1 maintained excess ammonium molybdate and removed completely any interference previously observed with ATP. As a
570
SEDDON
0
AND
FYNN
03
0.6
FIG. 2. Calibration curve of OD,., against Pi content using modified FiskeSubbaRow assay procedure. 0, P, estimation using 20 mM ammonium molybdate (1.6 mM final concentration). 0, PI estimation using 40 rnM (3.2 mM final concentration) ammonium molybdate. A, P, estimation using 40 mM ammonium molybdete (3.2 mM final concentration) in the presence of 10 pmoles ATP.
precautionary measure, systems with added HClok (ATPase assays terminat,ed with HCIO,) were allowed to stand 10 min at O”C, and the HClO,-treated sample neutralised by addition of NaOH before estimations of the Pi content were made using the above modified Fiske-SubbaRow method. (Nonenzymatic acid hydrolysis of ATP due to the lo-min incubation period in HClO, was found to be of the order of 0.3-0.4s and was corrected for). By this procedure the pH of the assay system, which critically affects ATP interference, was maintained constant throughout. REFERENCES KUSHMERICK, J. M. (1972) Anal. Biochem. 46, 129. BLUM, J. J., AND CHAMBERS, R. W. (1955) Biochim. Biophys. Acta 18, 601. WEIL-MALHERBE, H., ANTI GREEN, R. H. (1951) B&hem. J. 49, 286. FLYNN, R. M., JONES, M. E., AND LIPMANN, F. (1954) .7. Biol. Chem. 211, 791. BERENBLUM, I., AND CHAIN, E. (1938) Biochem. J. 32, 286.
1. 2. 3. 4. 5. 6. FISKE, C. H., 7. ALBERTY, R.
AND SUBBAROW,
A., SMITH,
Y.
(1925)
R. M., AND BOCK,
J. Biol.
Chem.
66, 375.
R. M. (1951) J. Biol.
Chem.
193,
425.
56, 566-570 (1973)
BIOCHEMISTRY
Orthophosphate Analysis by the Fiske-SubbaRow and Interference by Adenosine Phosphates Pyrophosphate
at Variable
B. SEDDON’ institute
Department of Science
of
Biochemistry,
and Technology,
Received
March
AND
Acid
Method and
pH
G. H. FYNN
University Manchester
of Manchester, M6’0 l&D, United
2, 1973; accepted
Kingdom
June 28, 1973
ATP, ADP, AMP, and pyrophosphate interfere with colour development in the Fiske-SubbaRow assay procedure. The extent of interference observed is dependent on the pH of the final assay system; small changes in pH at low pH values lead to large variations in the amount of ATP interference. Addition of excess ammonium molybdate and neutralisation of any previous acid additions (such that the pH is maintained constant throughout all assay procedures) removes interference and Pi can be estimated in the presence of relatively high ATP concentrations. In previous reports containing compounds
(14) form
it has been noted that various phosphatecomplexes with molybdic acid. In a recent
communication (1) Kushmerick has reported upon the interference with Pi analysis by ATP, AMP, pyrophosphate, and phosphorylcreatine when using the method of Berenblum and Chain (5) and the removal of such interferences by adding extra molyb’date. Previously Blum and Chambers (‘2) had reported on the interference by ATP, ADP, and ITP in the FiskeSubbaRow analysis and the removal of such interference by addition of extra molybdate. The interference was due to the formation of a colourless complex between ATP and molybdate which did not become coloured upon the addition of reducing agent and catalyst. During investigations of bacterial ATPase systems we have also observed interference with Pi analysis by ATP, ADP, and AMP, and pyrophosphate using the Fiske-SubbaRow assay procedure. While the removal of such interference by addition of extra molybdate has already been reported (1,2) we noticed that, with the Fiske-Subb8aRow method, such factors as pH and the type and phosphate content of the phosphatecontaining
compound
influenced
the extent
’ Present address: Department of Developmental Marischal College, Aberdeen, AB9 lAS, U.K. 566 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
of interference
obtained
Biology, University
with
of Aberdeen,
ORTHOPHOSPHATE
ANALYSIS
567
this assay system. Conditions (which are reported here) were adopted such that assay of ATPase activity by measuring the release of Pi could be monitored even with concent,rat,ions of ATP which interfered with the original Fiske-SubbaRow assay system. METHODS
Inorganic orthophosphate (Pi) was measured by the Fiske-SubbaRow procedure (6). The assay system contained O-4.4 ml sample; 0.4 ml 20 mM (or 40 mM, see Result’s section) ammonium molybdate in 5 P; H&30, ; 0.2 ml 1-amino-2-napthol-4-sulphonic acid ( ANS) in bisulphite solution (6)) and distilled water to a total volume of 5.0 ml. The colour was estimated at 660 nm after 17-min development using a Unicam SP 600 spectrophotometer with a lightpath of 1 cm. In experiments in which the effect of ATP was studied ATP (disodium salt,, Sigma Chemical Co.) at the concentration given in the Results section, was also added. The effects of ADP, AMP, (Boehringer Co. Ltd.), pyrophosphate, and adenosine were investigated by replacing ATP with the above components. During the investigations of bacterial ATPase activity enzyme-catalysed reactions were stopped by the addition of an equal volume of HClO, (0.7 M). In order t’o mimic these conditions Pi assays in t,he presence and absence of ATP were performed with HClO, added. Assays were also made on test systems where the added HClO, was neut,ralised by addition of NaOH. In all assay procedures tubes were allowed to stand for 10 min at’ 0°C after addition of HClO, (release of Pi by acid hydrolysis of ATP by this treatment was tested for by including incubations with ATP alone). Pi content was then estimated by the method of Fiske-SubbaRow (6) as described above. All values quoted in the results section refer to the concentration of components in the final 5.0-ml assay system unless stated otherwise. RESULTS
AND
DISCUSSION
Initial studies estimating Pi content in the presence of ATP gave variable values for Pi content. It soon became apparent that this was a result of pH variance since the lowest values obtained for Pi in the presence of a fixed amount of ATP were those obtained using largervolume aliquots (approaching 4.4 ml) of HClO,-treated samples. Higher values for Pi were obtained with low-volume aliquots of HClO,-t,reated samples and the highest values of all were obtained when HClO, had subsequently been neutralised with NaOH. Figure 1 shows the estimation of a fixed amount of Pi with different, ATP content (i) in the absence of HClO,, (ii) in the presence of HClO, (0.28 M), and (iii) in the
SEDDON AND FYNN
568
00
L
,, 4
AT&I)
FIG. 1. The effect of ATP concentratron and acid content on colour development in the Fiske-SubbaRow estimation of inorganic phosphate. The standard assay system contained Pi, 0.5 amoles and was assayed by the Fiske-SubbaRow procedure as described in the Methods section. ATP concentration was varied as shown in the figure. Assay procedure; 0, in the p r esence of HClO, (0.28 M) ; 0, in the absence of HCIOa; A, added HClO, neutralised with NaOH before assay; ---, assay in the absence of ATP with HClO, (0.28 M) added.
presence of HClO, neutralised with NaOH. It can be seen that interference of Pi determination increased with increasing concentration of ATP and that this interference was more pronounced in the presence of HClO,. Additions of HClO, concentrations up to 0.31 M did not interfere with the Pi assay test system when ATP was absent. The presence of HClO, resulted in a lowering of pH of the test system (pH of test system approximately 1.0-1.3). It is this decrease in pH which increases the interference of Pi assay brought about by ATP since neutralisation of HClO, reduced the interference of ATP to values similar to those observed in the absence of HCIO, (Fig. 11. The amount of ammonium molybdate present in the final test system is approximately 8 pmoles. At high and low pH values 16 pmoles and 12 pmoles ATP, respectively, completely abolished any “molybdenum blue” formation (Fig. 1). Thus, ammonium molybdate:ATP ratios 1:2 and 1: 1.5 effectively removed free ammonium molybdate from the test systems. Alberty, Smith, and Bock (71 have discussed the pk values of the phosphate groups of ATP and it is noticeable from their work that the primary phosphate groups have a pk value <2.0. Changes in pH therefore, at values <2.0 would influence the balance of ionised to nonionised
primary
phosphate
groups
and
thus
such groups could interfere with the assay of Pi.
the
extent
to
which
ORTHOPHOSPHATE
The
Additions
Effects
of Various
to assay system
Compounds
TABLE 1 on the Fiske-SubbaRow
of 0.5 pmoles
Concn
of additive 5.0 ml
Pi
ATP ATP ATP + HClOd ATP + ammonium ADP AMP Pyrophosphate Adenosine a The Fiske-SubbaRow using 20 mM ammonium in the table above.
569
ANALYSIS
1
Assay
in final
of Pi” y0 Inhibition of colour development at 660 nm
3rnM
0 100 20 0 50
3rnM 3rnM
12 100
4rnM
0
rnM
3rnM
molybdate
assay procedure molybdate (final
1 mM + 0.28 rnM 3rnM$3.2rnM
was as described in the Methods concentration 1.6 mM) except where
se&ion stated
Further studies with ADP, AMP, pyrophosphate, and adenosine (Table 1) indicated that the presence of an ionisable phosphate group was, in fact, responsible for the interference observed. The greater the number of ionisable phosphate groups present the stronger was the interference observed. Aden,osine did not show any interfering properties. It is assumed, as has been reported previously (1,2), that the various phosphate-containing compounds are reacting with t-he ammonium molybdate through their ionisable phosphate groups to form colourless complexes, thus rendering the ammonium molybdate inaccessible for reaction with Pi. Table 1 shows that increasing the final concentration of ammonium molybdate from 1.6 rnM to 3.2 rnM completely removed the interference due to ATP. Increasing the final concentration of ANS reagent did not restore “molybdenum blue” formation. As a result of the above findings and the possibility that samples for Pi assay may contain high concentrations of ATP, 3.2 mM final concentration of ammonium molybdate was used in the assay system for Pi estimation. Figure 2 shows the calibration curves for assays containing (i) 1.6 rnM ammonium molybdate (ii) 3.2 rnM ammonium molybdate and (iii) 3.2 mM ammonium molybdate in the presence of 10 @moles ATP. The modification employed did not alter the sensitivity nor the characteristics of the calibration curve. Increasing the concentration of ammonium molybdate such that the ammonium molybdate: ATP ratio is 5 2: 1 maintained excess ammonium molybdate and removed completely any interference previously observed with ATP. As a
570
SEDDON
0
AND
FYNN
03
0.6
FIG. 2. Calibration curve of OD,., against Pi content using modified FiskeSubbaRow assay procedure. 0, P, estimation using 20 mM ammonium molybdate (1.6 mM final concentration). 0, PI estimation using 40 rnM (3.2 mM final concentration) ammonium molybdate. A, P, estimation using 40 mM ammonium molybdete (3.2 mM final concentration) in the presence of 10 pmoles ATP.
precautionary measure, systems with added HClok (ATPase assays terminat,ed with HCIO,) were allowed to stand 10 min at O”C, and the HClO,-treated sample neutralised by addition of NaOH before estimations of the Pi content were made using the above modified Fiske-SubbaRow method. (Nonenzymatic acid hydrolysis of ATP due to the lo-min incubation period in HClO, was found to be of the order of 0.3-0.4s and was corrected for). By this procedure the pH of the assay system, which critically affects ATP interference, was maintained constant throughout. REFERENCES KUSHMERICK, J. M. (1972) Anal. Biochem. 46, 129. BLUM, J. J., AND CHAMBERS, R. W. (1955) Biochim. Biophys. Acta 18, 601. WEIL-MALHERBE, H., ANTI GREEN, R. H. (1951) B&hem. J. 49, 286. FLYNN, R. M., JONES, M. E., AND LIPMANN, F. (1954) .7. Biol. Chem. 211, 791. BERENBLUM, I., AND CHAIN, E. (1938) Biochem. J. 32, 286.
1. 2. 3. 4. 5. 6. FISKE, C. H., 7. ALBERTY, R.
AND SUBBAROW,
A., SMITH,
Y.
(1925)
R. M., AND BOCK,
J. Biol.
Chem.
66, 375.
R. M. (1951) J. Biol.
Chem.
193,
425.