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Interference with orthophosphate analysis by ATP and phosphorylcreatine
ANALYTICAL
46,
BIOCHEMISTRY
129-134
(1972)
Interference with Orthophosphate by ATP and Phosphorylcreatine MARTIN Department
of Physiology,
Harvard
Analysis
J. KUSHMERICK Medical
School, Boston, Massachusetts
02’115
Received June 25, 1971
The method of Berenblum and Chain (1) for analysis of orthophosphate (Pi) and subsequent modifications of this general method (2-4) are valuable and widely used for analysis in the presence of labile phosphoryl compounds and for separation of orthophosphate from mixtures. The principle of the method is the formation of a phosphoric-dodecamolybdic acid complex which can be selectively and rapidly extracted into an organic solvent phase. Its concentration can be measured directly (An,, = 310 nm; a, = 24 X 10e3liter mole-l cm-l) or after reduction to “phosphomolybdenum blue.” While testing the method of Wahler and Wollenberger (4) it was noted that the measured absorbance in analyses of mixtures containing known amounts of Pi was reduced when the mixtures contained large amounts of phosphorylcreatine (PCr) or ATP. The reason for this effect was studied. The results obtained show that both ATP and PCr can form complexes with molybdic acid to give falsely low estimates of the Pi content of mixtures and tissue extracts when these contain ATP or PCr in large molar excess over Pi. If recognized, the interference is easily overcome by adding extra molybdate so it is present in excess. This report extends the list of compounds known to form complexes with molybdic acid (1,5). In addition the rates of hydrolysis of ATP and PCr were measured under the conditions of the Wahler and Wollenberger method of analysis (4). Only PCr was hydrolyzed, at a low but not negligible rate; this hydrolysis had not been emphasized previously. METHODS
The det.ailed methods of Wahler and Wollenberger (4) were followed using reagent grade chemicals and glass-distilled water. Two precautions must be noted. First, certain lots of perchloric acid gave high reagent blank readings presumably because of trace contamination by phosphoric acid. Second, all samples of isopropyl acetate tested contained impurities which had significant absorbance at 310 nm. These were 129 @ 1972 hp Academic
Press. Inr.
130
MARTIN
J.
KUSHMERICP
removed satisfactorily by distillation, first 20% and last 10% of the starting volume being discarded. The distillate had an absorbance at 310 nm, 10 mm light path of less than 0.015 against distilled water. The analyses were done in 10 ml stoppered tubes (precooled at O’C) containing 4 ml isopropyl acetate and 2 ml sodium molybdate at various concentrations (see “Results”). The sample was added (1 ml), then 1 ml of 0.5 M HClO, and the analysis tubes were mixed immediately on a vortex mixer. The tubes were insulated from room temperature air by foam urethan jackets; after 60 set mixing the contents had warmed to 6”. Brief centrifugation speeded the separation of phases so that the uppermost s/4 of the isopropyl acetate was removed within 2 to 3 min after addition of the sample to the analysis tube. RESULTS
Preliminary experiments with orthophosphate (Pi) alone indicated that the rate of extraction of phosphomolybdic acid into the isopropyl acetate phase increased at higher concentrations of molybdate. Using a concentration of 3.75 mM sodium molybdate in the analysis tube (15 pmole per tube) as recommended by Wahler and Wollenberger, 71% of the final absorbance was obtained after 20 see of mixing and 88% after 40 seeof mixing. When the molybdate concentration was increased 5-fold, 91% of the final absorbance was reached after 20 see of mixing. With both concentrations of molybdate complete extraction of phosphopolymolybdic acid was achieved by 60 see of mixing because no additional chromogen was extracted during an additional 60 set period of mixing. Tests of the efficiency and rapidity of extraction were made by premixing Pi, sodium molybdate (3.75 mM in analysis tube), and perchloric acid to form phosphomolybdic acid, then adding isopropyl acetate and mixing. 95% of the final absorbance was reached after 20 set of mixing. These results show that extraction of the chromogen is not rate limiting and that increasing sodium molybdate concentration in the analysis tube increased the rate of formation of phosphomolybdic acid. These results are consistent with a kinetic study of phosphomolybdenum blue formation made in nitric and sulfuric acid (6). Analyses in duplicate were made using various concentrations of sodium molybdate in the presence of both Pi and PCr; each analysis tube was mixed for 60 sec. Several features of the results are illustrated in Fig. 1. The concentration of sodium molybdate (3.75 mM) in the analysis tube originally suggested (4) is just the minimum necessary to effect complete formation and extraction of the complex within 60 set when only Pi is present. In the case of Pi with a 20-fold molar excess of PCr (stock solution of the latter contained Pi or was partly hydro-
ORTHOPHOSPHATE
ANALYSIS
131
FIG. 1. Absorbance (10 mm, h = 310 nm) of isopropyl acetate after carrying out analysis as described in the presence of different amounts of sodium molybdate. Analysis tube contained 1 ml aqueous sample, 1 ml 0.5M HC101, 2 ml sodium molybdate, and 4 ml isopropyl acetate. (0) Blank; (A) 5 X 10e8 mole orthophosphate added; ( n ) 1 X lOme mole phosphorylcreatine added; (X) 5 X 10“ mole orthophosphate and 1 X lo-” mole phosphorylcreatine added.
lyzed-see below), higher concentrations of molybdate were necessary to form and extract the phosphomolybdic acid. The absorbance of the reagent blank increased very slightly over the range of molybdate concentrations tested, confirming the low partition coefficient of the various polymoIybdic acids in isopropyl acetate. Creatine did not interfere when tested up to 27-fold molar excess over Pi using 3.75 mM sodium molyddate in the analysis tube. In a similar way the effects of ATP, AMP, and pyrophosphate on the Pi analysis were tested. The results (Fig. 2) show that additional molybdate was required for complete formation and extraction of phosphomolybdic acid in the presence of 100-fold molar excess of ATP. Qualitatively the same result was obtained using a 27-fold molar excess of ATP but the absorbance curve of Pi plus ATP was shifted to the left. AMP and pyrophosphate interference was just detectable using a 20-fold molar excess of each over Pi. Interference by ATP in the Fiske-SubbaRow analysis has been reported (7). The following tests were made to measure the extent of hydrolysis of PCr and ATP during analysis using 18.8 rnnl sodium molybdate (75 pmoles in each analysis tube). PCr or ATP, sodium molybdate, and perchloric acid were mixed in an analysis tube and stored at 0°C for various times, after which isopropyl acetate (0”) was added and the rest of the analysis carried out as usual. In Fig. 3 the slope is the hydrol-
132
MARTIN
J.
XUSHMERICK
l
i(
ATP l
Amount
of
Sodium
Molybdate
Present
(pmoies INIOWSIS tube)
FIG. 2. Absorbance (10 mm, h=310 nm) of isopropyl acetate after carrying out analysis as described in the presence of different amounts of sodium molybdate. Contents of analysis tube as described in Fig. 1. (0) Blank; (A) 5 X lad mole orthophosphate added; (m) 5 X 10’ mole ATP added ; (X) 5 X lo-* mole orthophosphate and 5 X lo-’ mole ATP added.
1
L
0
5 TIME
IO
15
(min)
FIG. 3. Hydrolysis of phosphorylcreatine under conditions of orthophosphate analysis, expressed as fraction of PCr added. Contents of analysis tube ss described in Fig. 1 using 75 amoles sodium molybdate per tube. (0) 7 X 10” mole PCr added; (X) 3.7 X lo-’ mole PCr added; (0) creatine present in PCr stock solution by direct analysis (8) which equals orthophosphate contamination if sample of PCr was initially 100%PG.
ORTHOPHOSPHATE
ANALYSIS
133
ysis rate of PCr; the intercept is the Pi contamination in the stock PCr solution plus that produced by hydrolysis during the 60 set of mixing. The rate of PCr hydrolysis averaged 1.6%/min. In a similar test ATP hydrolysis was not detected. DISCUSSION
Since in all cases of interference tested the absorbance reached a maximal value by increasing the amount of molybdate in the analysis tube, it seems likely the interference is due to formation of complexes with molybdic acid in competition with Pi. Examination of the molar ratios relative to the amount of Pi present suggests that the ability to form such complexes decreases in the order: PCr > ATP > AMP N pyrophosphate. Approximately 15 to 20 pmoles of extra sodium molybdate were necessary to overcome the inhibition by 1 Fmole of PCr (Fig. 2) ; this suggests that 15 to 20 moles of molybdic acid monomer bind per mole of PCr. By a similar estimation (Fig. 2), 5 moles of molybdic acid monomer bind per mole of ATP. Previously no hydrolysis of PCr was detected using the analysis of Wahler and Wollenberger (4), which was designed to minimize hydrolysis of labile phosphoryl compounds by its speed and low temperature. The measurable rate of PCr hydrolysis during the assay is low enough to be disregarded only when the concentration of PCr is similar to that of Pi being measured; otherwise the measured Pi has a significant contribution from hydrolyzed PCr. In the case of skeletal muscle extracts the molar ratio of PCr to Pi is in the range of 2O:l. Thus measured “free” orthophosphate, using the method of Wahler and Wollenberger (4)) may well be overestimated by about 30%. The question of the true orthophosphate content of muscle has been considered (9). It is fortunate that many measurements of chemical change during muscle contraction have been obtained by difference between a control and experimental muscle since any error would apply equally to both. This is not true in cases in which there was a large change in PCr content. SUMMARY
1. Phosphorylcreatine and ATP can interfere with orthophosphate analysis by forming complexes with molybdic acid. Once recognized, this problem is overcome by adding extra molybdate t.o the analysis mixture so that this reagent is present in sufficient excess. 2. Phosphorylcreatine was found to be hydrolyzed at a low but measurable rate during the rapid analysis at 0°C described by Wahler and Wollenberger,
134
MARTIN
J.
KUSHMERICK
ACKNOWLEDGMENTS This work was carried out Exchange Fellowship from the ment of Physiology, University ful assistance and Professor D.
largely during the tenure of a British-American American Heart Association held in the DepartCollege London. I thank Claude Gilbert for skillR. Wilkie for laboratory accommodations. REFERENCES
1. BERENBLUM, I., AND CHAIN, E., Biochem. J. 32, 286 (1938). 2. MARSH, B. B., Biochim. Biophys. Acta 32, 357 (1959). 3. MARTIN, J. B., AND DOTY, D. M., Anal. Chem. 21, 965 (1949). 4. WAHLER, B. E., AND WOLLENBERGER, A., Biochem. 2. 329, 508 (1958). 5. VREMAN, H. J., AND J~BSIS, F. F., Anal. Biochem. 17, 108 (1966). 6. CROUCH, S. R., AND MALMSTADT, H. V., Anal. Chem. 39, 1084 (1967). 7. BLUM, J. J., AND CHAMBERS, R. W., Biochim. Biophys. Acta 18, 601 (1955). 8. ENNOR, A. H., in “Methods in Enzymology” (S. P. Colowick and N. D. Kaplan, eds.), Vol. III, p. 850. Academic Press, New York, 1957. 9. SERAPDARIAN, K., MOMMAERTS, W. F. H. M., WALLNER, A., AND GUILLORY, R. J., J. Biol. Chem. 236, 2071 (1961).
46,
BIOCHEMISTRY
129-134
(1972)
Interference with Orthophosphate by ATP and Phosphorylcreatine MARTIN Department
of Physiology,
Harvard
Analysis
J. KUSHMERICK Medical
School, Boston, Massachusetts
02’115
Received June 25, 1971
The method of Berenblum and Chain (1) for analysis of orthophosphate (Pi) and subsequent modifications of this general method (2-4) are valuable and widely used for analysis in the presence of labile phosphoryl compounds and for separation of orthophosphate from mixtures. The principle of the method is the formation of a phosphoric-dodecamolybdic acid complex which can be selectively and rapidly extracted into an organic solvent phase. Its concentration can be measured directly (An,, = 310 nm; a, = 24 X 10e3liter mole-l cm-l) or after reduction to “phosphomolybdenum blue.” While testing the method of Wahler and Wollenberger (4) it was noted that the measured absorbance in analyses of mixtures containing known amounts of Pi was reduced when the mixtures contained large amounts of phosphorylcreatine (PCr) or ATP. The reason for this effect was studied. The results obtained show that both ATP and PCr can form complexes with molybdic acid to give falsely low estimates of the Pi content of mixtures and tissue extracts when these contain ATP or PCr in large molar excess over Pi. If recognized, the interference is easily overcome by adding extra molybdate so it is present in excess. This report extends the list of compounds known to form complexes with molybdic acid (1,5). In addition the rates of hydrolysis of ATP and PCr were measured under the conditions of the Wahler and Wollenberger method of analysis (4). Only PCr was hydrolyzed, at a low but not negligible rate; this hydrolysis had not been emphasized previously. METHODS
The det.ailed methods of Wahler and Wollenberger (4) were followed using reagent grade chemicals and glass-distilled water. Two precautions must be noted. First, certain lots of perchloric acid gave high reagent blank readings presumably because of trace contamination by phosphoric acid. Second, all samples of isopropyl acetate tested contained impurities which had significant absorbance at 310 nm. These were 129 @ 1972 hp Academic
Press. Inr.
130
MARTIN
J.
KUSHMERICP
removed satisfactorily by distillation, first 20% and last 10% of the starting volume being discarded. The distillate had an absorbance at 310 nm, 10 mm light path of less than 0.015 against distilled water. The analyses were done in 10 ml stoppered tubes (precooled at O’C) containing 4 ml isopropyl acetate and 2 ml sodium molybdate at various concentrations (see “Results”). The sample was added (1 ml), then 1 ml of 0.5 M HClO, and the analysis tubes were mixed immediately on a vortex mixer. The tubes were insulated from room temperature air by foam urethan jackets; after 60 set mixing the contents had warmed to 6”. Brief centrifugation speeded the separation of phases so that the uppermost s/4 of the isopropyl acetate was removed within 2 to 3 min after addition of the sample to the analysis tube. RESULTS
Preliminary experiments with orthophosphate (Pi) alone indicated that the rate of extraction of phosphomolybdic acid into the isopropyl acetate phase increased at higher concentrations of molybdate. Using a concentration of 3.75 mM sodium molybdate in the analysis tube (15 pmole per tube) as recommended by Wahler and Wollenberger, 71% of the final absorbance was obtained after 20 see of mixing and 88% after 40 seeof mixing. When the molybdate concentration was increased 5-fold, 91% of the final absorbance was reached after 20 see of mixing. With both concentrations of molybdate complete extraction of phosphopolymolybdic acid was achieved by 60 see of mixing because no additional chromogen was extracted during an additional 60 set period of mixing. Tests of the efficiency and rapidity of extraction were made by premixing Pi, sodium molybdate (3.75 mM in analysis tube), and perchloric acid to form phosphomolybdic acid, then adding isopropyl acetate and mixing. 95% of the final absorbance was reached after 20 set of mixing. These results show that extraction of the chromogen is not rate limiting and that increasing sodium molybdate concentration in the analysis tube increased the rate of formation of phosphomolybdic acid. These results are consistent with a kinetic study of phosphomolybdenum blue formation made in nitric and sulfuric acid (6). Analyses in duplicate were made using various concentrations of sodium molybdate in the presence of both Pi and PCr; each analysis tube was mixed for 60 sec. Several features of the results are illustrated in Fig. 1. The concentration of sodium molybdate (3.75 mM) in the analysis tube originally suggested (4) is just the minimum necessary to effect complete formation and extraction of the complex within 60 set when only Pi is present. In the case of Pi with a 20-fold molar excess of PCr (stock solution of the latter contained Pi or was partly hydro-
ORTHOPHOSPHATE
ANALYSIS
131
FIG. 1. Absorbance (10 mm, h = 310 nm) of isopropyl acetate after carrying out analysis as described in the presence of different amounts of sodium molybdate. Analysis tube contained 1 ml aqueous sample, 1 ml 0.5M HC101, 2 ml sodium molybdate, and 4 ml isopropyl acetate. (0) Blank; (A) 5 X 10e8 mole orthophosphate added; ( n ) 1 X lOme mole phosphorylcreatine added; (X) 5 X 10“ mole orthophosphate and 1 X lo-” mole phosphorylcreatine added.
lyzed-see below), higher concentrations of molybdate were necessary to form and extract the phosphomolybdic acid. The absorbance of the reagent blank increased very slightly over the range of molybdate concentrations tested, confirming the low partition coefficient of the various polymoIybdic acids in isopropyl acetate. Creatine did not interfere when tested up to 27-fold molar excess over Pi using 3.75 mM sodium molyddate in the analysis tube. In a similar way the effects of ATP, AMP, and pyrophosphate on the Pi analysis were tested. The results (Fig. 2) show that additional molybdate was required for complete formation and extraction of phosphomolybdic acid in the presence of 100-fold molar excess of ATP. Qualitatively the same result was obtained using a 27-fold molar excess of ATP but the absorbance curve of Pi plus ATP was shifted to the left. AMP and pyrophosphate interference was just detectable using a 20-fold molar excess of each over Pi. Interference by ATP in the Fiske-SubbaRow analysis has been reported (7). The following tests were made to measure the extent of hydrolysis of PCr and ATP during analysis using 18.8 rnnl sodium molybdate (75 pmoles in each analysis tube). PCr or ATP, sodium molybdate, and perchloric acid were mixed in an analysis tube and stored at 0°C for various times, after which isopropyl acetate (0”) was added and the rest of the analysis carried out as usual. In Fig. 3 the slope is the hydrol-
132
MARTIN
J.
XUSHMERICK
l
i(
ATP l
Amount
of
Sodium
Molybdate
Present
(pmoies INIOWSIS tube)
FIG. 2. Absorbance (10 mm, h=310 nm) of isopropyl acetate after carrying out analysis as described in the presence of different amounts of sodium molybdate. Contents of analysis tube as described in Fig. 1. (0) Blank; (A) 5 X lad mole orthophosphate added; (m) 5 X 10’ mole ATP added ; (X) 5 X lo-* mole orthophosphate and 5 X lo-’ mole ATP added.
1
L
0
5 TIME
IO
15
(min)
FIG. 3. Hydrolysis of phosphorylcreatine under conditions of orthophosphate analysis, expressed as fraction of PCr added. Contents of analysis tube ss described in Fig. 1 using 75 amoles sodium molybdate per tube. (0) 7 X 10” mole PCr added; (X) 3.7 X lo-’ mole PCr added; (0) creatine present in PCr stock solution by direct analysis (8) which equals orthophosphate contamination if sample of PCr was initially 100%PG.
ORTHOPHOSPHATE
ANALYSIS
133
ysis rate of PCr; the intercept is the Pi contamination in the stock PCr solution plus that produced by hydrolysis during the 60 set of mixing. The rate of PCr hydrolysis averaged 1.6%/min. In a similar test ATP hydrolysis was not detected. DISCUSSION
Since in all cases of interference tested the absorbance reached a maximal value by increasing the amount of molybdate in the analysis tube, it seems likely the interference is due to formation of complexes with molybdic acid in competition with Pi. Examination of the molar ratios relative to the amount of Pi present suggests that the ability to form such complexes decreases in the order: PCr > ATP > AMP N pyrophosphate. Approximately 15 to 20 pmoles of extra sodium molybdate were necessary to overcome the inhibition by 1 Fmole of PCr (Fig. 2) ; this suggests that 15 to 20 moles of molybdic acid monomer bind per mole of PCr. By a similar estimation (Fig. 2), 5 moles of molybdic acid monomer bind per mole of ATP. Previously no hydrolysis of PCr was detected using the analysis of Wahler and Wollenberger (4), which was designed to minimize hydrolysis of labile phosphoryl compounds by its speed and low temperature. The measurable rate of PCr hydrolysis during the assay is low enough to be disregarded only when the concentration of PCr is similar to that of Pi being measured; otherwise the measured Pi has a significant contribution from hydrolyzed PCr. In the case of skeletal muscle extracts the molar ratio of PCr to Pi is in the range of 2O:l. Thus measured “free” orthophosphate, using the method of Wahler and Wollenberger (4)) may well be overestimated by about 30%. The question of the true orthophosphate content of muscle has been considered (9). It is fortunate that many measurements of chemical change during muscle contraction have been obtained by difference between a control and experimental muscle since any error would apply equally to both. This is not true in cases in which there was a large change in PCr content. SUMMARY
1. Phosphorylcreatine and ATP can interfere with orthophosphate analysis by forming complexes with molybdic acid. Once recognized, this problem is overcome by adding extra molybdate t.o the analysis mixture so that this reagent is present in sufficient excess. 2. Phosphorylcreatine was found to be hydrolyzed at a low but measurable rate during the rapid analysis at 0°C described by Wahler and Wollenberger,
134
MARTIN
J.
KUSHMERICK
ACKNOWLEDGMENTS This work was carried out Exchange Fellowship from the ment of Physiology, University ful assistance and Professor D.
largely during the tenure of a British-American American Heart Association held in the DepartCollege London. I thank Claude Gilbert for skillR. Wilkie for laboratory accommodations. REFERENCES
1. BERENBLUM, I., AND CHAIN, E., Biochem. J. 32, 286 (1938). 2. MARSH, B. B., Biochim. Biophys. Acta 32, 357 (1959). 3. MARTIN, J. B., AND DOTY, D. M., Anal. Chem. 21, 965 (1949). 4. WAHLER, B. E., AND WOLLENBERGER, A., Biochem. 2. 329, 508 (1958). 5. VREMAN, H. J., AND J~BSIS, F. F., Anal. Biochem. 17, 108 (1966). 6. CROUCH, S. R., AND MALMSTADT, H. V., Anal. Chem. 39, 1084 (1967). 7. BLUM, J. J., AND CHAMBERS, R. W., Biochim. Biophys. Acta 18, 601 (1955). 8. ENNOR, A. H., in “Methods in Enzymology” (S. P. Colowick and N. D. Kaplan, eds.), Vol. III, p. 850. Academic Press, New York, 1957. 9. SERAPDARIAN, K., MOMMAERTS, W. F. H. M., WALLNER, A., AND GUILLORY, R. J., J. Biol. Chem. 236, 2071 (1961).