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Determination of inorganic phosphate in the presence of labile organic phosphates
ANALYTICAL
BICCHEMISTBY
Determination
27, 65-72 (1969)
of Inorganic Phosphate in the Presence of Labile Organic Phosphates
R. PARVIN
AND
ROBERTS
A. SMITH
The Biochemistry Divkion, Department of Chemistry, University Calijorniu, Los Angeles, California 900.94
of
Received March 26, 1968
A number of modifications of the Fiske and SubbaRow (1) procedure for the determination of orthophosphate in the presence of labile phosphate compounds have appeared. The altered procedures usually involve organic solvent extraction of the phosphomoIybdate complex (2, 3) or formation of the complex at higher pH values (4) to minimize the molybdate-catalyzed acid hydrolysis of labile phosphates (5). The limitations of these methods have been pointed out by Marsh (6) in the presentation of a highly useful and sensitive procedure. However in our hands, Marsh’s method gave erroneous results in kinetic analyses of phosphatases when large numbers of samples were handled, presumably due to the chelation of molybdate (7) and the resulting instability of the phosphomolybdate complex in the presence of citrate (6). Phosphorus estimation as molybdovanadophosphate complex was introduced by Misson (8) and has been used in different forms for phosphorus determination in steel and ores (see 9). Michelsen’s modification of this method (10) is sensitive and requires only a low concentration of acid and molybdate. Thus, it appeared promising for phosphate determinations in the presence of labile phosphate compounds. By making use of the solubility of molybdovanadophosphoric acid in butanol we have developed a rapid procedure which largely avoids interference due to the hydrolysis of labile phosphates and yields a stable complex. The details of this method are outlined here. MATERIALS
Chemicals. Glucose l-phosphate, glucose 6-phosphate, and fructose l&diphosphate were obtained from Calbiochem; ATP and creatme phosphate were Sigma products; other chemicals were Baker analyzed reagents. Reagents. These were much the same as described by Michelsen (10) 65
66
PARVIN
AND
SMITH
and were prepared as follows: (a) 10N hydrochloric acid. (5) 0.234% ammonium metavanadate-2.34 gm NI$VO, was dissolved in 500 ml of hot water, 28 ml of concentrated HCl was added, and the solution was made to 1 liter with distilled water after cooling. (c) 3.53% ammonium molybdate35.3 gm of (NH,)M0,0244HZ0 was dissolved in warm water and diluted to 1 liter. Ammonium nsetavccnudate-mol$date reagent (reagent I). To about 150 to 300 ml water, 14 ml of (a) and 10 ml of reagent (5) were added and mixed; finally 20 ml of reagent (c) was added and the volume made up to 1 liter with distilled water. PROCEDURE Samples containing from 10 to 150 meoles of orthophosphate are diluted to 1.5 ml with water in 14 X 125 mm test tubes and 3.0 ml of n-butanol is added to all the tubes. This is followed by addition of 1.5 ml of ammonium metavanadate-molybdate (reagent I) and the tube is immediately shaken for 6 to 10 set either by hand or on a Vortex mixer. The absorbancy of the clear butanol layer obtained after standing for a few minutes .or ,after brief centrifugation is measured at 310 my against a reagent. blank. If the butanol layer is turbid it is clarified by the addition of 0.05 ml of methanol before recording the absorbancy. As long as the relative volumes of the orthophosphate solution, butanol, and reagent I are kept in the ratio of 1: 2 : 1, any suitable volume may be chosen. RESULTS In several experimental procedures requiring the determination of orthophospbate in the presence of labile phosphates, the phosphomolybdate complex is usually extracted in a suitable organic solvent to. avoid the error ,due td molybdate-catalyzed acid hydrolysis of labile phosphates (6). The possibility of employing solvent extraction of the molybdovanadophosphoric acid complex formed in Michelsen’s procedure (10) was explored. When n-butanol was used for the extraction of the molybdovanadophosphate complex, formed under the conditions of Michelsen (10) using reagent I of acid strength up to ‘0.08 N, extraction of the complex was incomplete. However, subsequent experiments showed that if the butanol used for the extraction of the molybdovanadophosphate complex was acidified (0.6 ml of 0.1 N HCl to 100 ml of butanol) the complex could be completely extracted. The need for acidification of the butanol prior to extraction of the complex was obviated if the ‘acid strength of reagent I was increased to at least 0.12 N. When the orthophosphate color was developed in this way, an identical extinction, coefficient for the pliosphomolybdovanadate complex was found in the
INORGANIC
PHOSPHATE
67
DETERMINATION
aqueous and in the butanol method. The absorption spectrum of the molybdovanadophosphoric acid complex in butanol showed (Fig. 1) a. peak at 310 my, which was slightly lower than the reported maximum absorption of 315 rnp in water (10). Since in the Michelsen method variation in the acid concentration affected the complex formation as well
1
290
3;0
330 WAVELENGTH
350 rnp
FIQ. 1. Absorption spectra of molybdovanadophosphoric in butanol. Details were aa described under “‘Procedure.” reagent blank was measured against butaaol. Curves 2 and dovanadophoaphate derived from 0.1 rmole Pi obtained measured against reagent blank and butanol, respectively.
370
acid and reagent blanks Curve 1, absorption of 3, absorbance of molybby the present method
as the blank absorption, the effect of varying acidities on the butanol extraction procedure was determined and the results are shown in Figure 2. As is evident from these results the final acid strength could, be varied between 0.06 and 0.15 N (with respect to the aqueous phase) without appreciably affecting either the blank absorption or the color development in the butanol phase. Thus, unlike the method of Michelsen (lo),
68
PARVIN
AND
SMITH
the butanol extraction procedure allows a wider range of acidities. A standard plot for orthophosphate (Fig. 3) using the butanol extraction procedure showed that Beer’s law is followed for up to at least 166 mqoles of orthophosphate. The color in the butanol phase remained unchanged for up to 26 hr at room temperature. Phosphorus determination in the presence of labile phosphate esters. Samples of fructose 1,6-diphosphate, ATP, glucose l-phosphate, and 0.0
20.6-
X
0 Gi ta
yj 0.4
X
-
-x-
,/x-x-x-x
I
X
x-x-xwx ‘X
/
/
/
: E
0
WI zoz-
0.;2 FINAL
0.06 ACID
0.i NORMALITY
0.;4 f H20
Oh PHASE)
FIQ. 2. Effect of acid concentration on absorbance of reagent blank and phosphomolybdovanadate in butanol extraction procedure. To 0.1 smole Pr solution or water (for reagent blank) sufkient HCl was added to bring the final acidity to that indicated in the 3 ml aqueous layer. Volume was made up to 1.!5 ml with water; 3 ml butanol was added, followed by 1.5 ml of reagent I. Following extraction in butanol, phosphorue color (X) in butanol phase was measured at 310 rnp age&& corresponding reagent blank. Reagent blanks (0) were read against butanol shaken with equal volume of water.
creatine phosphate were analyzed for their orthophosphate content by the present method and compared with the results obtained by the method of Marsh (6). From the results presented in Table 1, it is seen that the orthophosphate contents of fructose 1,6-diphosphate, glucose l-phosphate, and ATP were similar as detected by the two methods, while for creatine phosphate the Marsh method gave nearly twice as much orthophosphate content as the present method. Since molybdate-catalyzed hydrolysis of certain labile phosphates is well known (4, 5), it appea,rs likely that the
INORGANIC
PHOSPHATE
DETERMINATION
Pi mp
FRI. 3. Standard
plot for orthophosphate.
moles
Details as described under “Procedure.”
higher molybdate concentration used in the Marsh method @S-fold that in the present method) was responsible for creatine phosphate hydrolysis. Although the Marsh method (6) makes use of citrate to chelate excess molybdate, the short contact of phosphate compounds with molybdate before citrate addition was sufhcient to hydrolyze very labile phosphates such as creatine phosphate (5). To check whether it was necessary to bind the excess molybdate in the present method, and to what extent the molybdate-catalyzed hydrolysis of labile phosphates in the aqueous phase contributes to error, several tubes containing phosphate compounds were subjected to the complete procedure except that the butanol layer remained unseparated from the aqueous phase for varying time intervals. When creatine phosphate was present, the increase in phosphate color in the butanol phase was 15-170/o in 1 hr. With glucose l-phosphate the color increase was never more than 1.5% in 1 hr. After separation of the aqueous and butanol layer, no further increase in the absorbance was seen in the butanol layer. Determination
TABLE 1 of Phosphate Contents of Labile Organic Phosphatea by the Marsh and bv the Present Method Molar
Labile
phosphates
Glucose l-phosphate Fructose 1,Bdiphoephate Creatine phosphate ATP
Present
per cent Pi content
method
2.81 1.40 3.25 4.9
&B obtained Marsh
by method
2.90 1.41 6.8 5.2
70
PARVIN
AND
SMITH
Interjering substances. Similar to other methods, the present procedure was also affected by the presence of higher amounts of citrate, ATP, and pyrophosphate. However, because of the marked sensitivity of the present procedure, it was possible to analyze the orthophosphate accurately in the presence of these inhibitory substances by taking smaller aliquots of samples representing no less than 0.01 mole of orthophosphate and no more than 1 pole of ADP, ATP, or citrate (0.5 eole in the case of pyrophosphate). Thus, orthophosphate could be determined by the present method despite a lOO-fold excess of citrate or ATP being present and interference is simply eliminated by taking smaller aliquots. The presence of small amounts of protein (up to 200 pg of rat liver supernatant protein or bovine serum albumin) did not interfere in the butanol extraction method provided the same amount of protein was present in the corresponding blank. At higher protein concentrations, part of the yellow colored complex remained absorbed on the precipitated protein and, therefore, protein precipitation prior to orthophosphate estimation was necessary. Trichloroacetic acid (up to 16 mg/ml) used for protein precipitation did not affect the final absorbance providing the blank received identical treatment. DISCUSSION The phosphomolybdovanadate complex formation method has been applied to total phosphorus determination in biological materials (11) as well as to the measurement of orthophosphate released during an adenosine triphosphatase reaction (12). In the latter case, due to simultaneous hydrolysis of ATP, it was required that absorbancies be measured at constant time intervals. The extraction of phosphomolybdovanadate in butanol under appropriate conditions markedly improved such a phosphorus estimation method. As shown by the present study, the interference due to the hydrolysis of labile phosphates was minimal compared to other procedures. Further, labile esters remain in contact with the acid phosphomolybdovanadate for only a short time. The color development is very rapid, requiring less than 2 min, and the complete extraction of the phosphomolybdovanadate complex in butanol occurs within a few seconds. Once color has been developed, subsequent contact between the butanol phase and the lower aqueous layer does not appreciably affect colors in the butanol phase when the labile phosphate compounds present are glucose l-phosphate, ATP, or fructose 1,8diphosphate. Molybdate-catalyzed acid hydrolysis of phosphate compounds appeared to have no effect in the butanol phase once the color had been developed and butanol extraction performed. Only when creatine phosphate, which is known to
INORGANIC
PHOSPHATE
DETERMINATION
71
be extremely acid-labile (5)) was present was it advantageous to separate the butanol layer immediately after color development. The sensitivity of the method and the stability of the color developed makes this procedure abundantly amenable to enzyme kinetic studies involving analysis of orthophosphate in the presence of labile phosphates. The stability of the colors in the presence of labile phosphates probably results from the removal of excess molybdate from the aqueous phase by the butanol extraction. Furthermore, the initial molybdate concentration is very low. Vanadate, which is also present in low concentrations, does not accelerate acid hydrolysis of phosphate esters (5). In general, the present butanol extraction procedure offers distinct advantages over the Michelsen method since the present procedure tolerates much wider range of final acidities (between 0.06 to 0.15). Further, in the present procedure the blank absorptions are not affected by small variations in the acidity. In the procedure for phosphorus determination described in the experimental section of this paper, the final acidity is 0.07 N, which was appropriate for the estimation of phosphate in the presence of labile phosphates; however, when phosphate samples are free from labile phosphates, any acid normality between 0.06 to 0.15 N may be chosen. Thus for phosphate determination in acid-digested samples, the acid strength of reagent I can be lowered to 0.042 N and a suitable aliquot of the aciddigested samples taken to give final acid normalities from 0.06 to 0.15. The phosphomolybdate vanadate complex formation, unlike phosphomolybdate formation (14, 15)) has been found to be free from interferences due to silicate, arsenate, and iron (13). The presence of excessive amounts of nucleoside di- and triphosphates, pyrophosphate, and citrate, which inhibit color development in methods based on phosphomolybdate formation (16-l@, also interferes in the presently described method. However, sensitivity of the present method allows analyses to be performed by taking suitable aliquots so as to keep the concentration of color inhibitory substances low. During several enzyme studies involving phosphate determination, the present procedure has proved extremely valuable because of the sensitivity and rapidity of the method. Since the presence of a small amount of protein does not interfere, addition of reagent I serves to terminate the reactions as well as to develop simultaneously the color that conveniently allows a large number of analyses to be performed with a great precision. SUMMARY
An operationally simple and rapid procedure of orthophosphate determination, based on the formation of a phosphomolybdovanadate complex,
72
PAWIN
AND SMITH
followed by its subsequent extraction into butanol, is described. The procedure is particularly advantageous for estimation of orthophosphate in the presence of labile phosphates, and yields stable colors. Conditions affecting the reliability of this procedure are evaluated. ACKNOWLEDGMENTS This work was supported in part by a grant from the United Statea Public Health Service (GM 13407) and by the Univemity of California Cancer Coordinating Committee. REFERENCES 1. FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 66, 375 (1925). 2. BERENBLUISS, I., AND CHAIN, E., Biochem. J. 32, 295 (19338). 3. MARTIN, J. B., AND DOTY, D. M., Anal. Chem. 21, 965 (1949). 4. LOWRY, 0. H., AND LOPEZ, J. A., J. Biol. Chem. 162, 421 (1946). 5. WEIL-MALHERBE, H., AND GREEN, R. H., Biochem. J. 49, 286 (1951). 6. MAIWH, B. B., B&him. Biophys. Acta 32, 357 (1959). 7. IMSANDE, J., AND EPHRUSSI, B., Science 144, 854 (1964). 8. MISSON, G., Chem. Ztg. 32, 633 (1908). 9. QUINLAN, K. P., DE SESA, M. A., Anal. Chem. 27, 1626 (1955). 10. MICHELSEN, 0. B., Anal. Chem. 29, 69 (1957). 11. KOENIQ, R. A., AND JOHNSON, C. R., Znd. Eng. Chem., Anal. Ed. 14, 155 (1942). 12. LECOCQ, J., AND INEST, G., Anal. B&hem. 15, 160 (1966). 13. KITSON, R. E., AND MELLON, M. G., Znd. Eng. Chem., Anal. Ed. 16, 379 (1944). 14. BOLTZ, D. F., AND MELLON, M. G., Anal. Chem. 19, 873 (1947). 15. WADELIN, C., AND MELLON, M. G., Anal. Chem. 25, 1668 (1953). 16. BLUM, J. J., AND CHAMBERS, R. W., Biochim. Biophys. Acta 18, 601 (1955). 17. R~R, G. W., J. Biol. Chsm. 230, 643 (1958). 18. D’ADAMO, JR., A. F., AND BROICH, J. R., Anal. Biochem. 4, 420 (1962).
BICCHEMISTBY
Determination
27, 65-72 (1969)
of Inorganic Phosphate in the Presence of Labile Organic Phosphates
R. PARVIN
AND
ROBERTS
A. SMITH
The Biochemistry Divkion, Department of Chemistry, University Calijorniu, Los Angeles, California 900.94
of
Received March 26, 1968
A number of modifications of the Fiske and SubbaRow (1) procedure for the determination of orthophosphate in the presence of labile phosphate compounds have appeared. The altered procedures usually involve organic solvent extraction of the phosphomoIybdate complex (2, 3) or formation of the complex at higher pH values (4) to minimize the molybdate-catalyzed acid hydrolysis of labile phosphates (5). The limitations of these methods have been pointed out by Marsh (6) in the presentation of a highly useful and sensitive procedure. However in our hands, Marsh’s method gave erroneous results in kinetic analyses of phosphatases when large numbers of samples were handled, presumably due to the chelation of molybdate (7) and the resulting instability of the phosphomolybdate complex in the presence of citrate (6). Phosphorus estimation as molybdovanadophosphate complex was introduced by Misson (8) and has been used in different forms for phosphorus determination in steel and ores (see 9). Michelsen’s modification of this method (10) is sensitive and requires only a low concentration of acid and molybdate. Thus, it appeared promising for phosphate determinations in the presence of labile phosphate compounds. By making use of the solubility of molybdovanadophosphoric acid in butanol we have developed a rapid procedure which largely avoids interference due to the hydrolysis of labile phosphates and yields a stable complex. The details of this method are outlined here. MATERIALS
Chemicals. Glucose l-phosphate, glucose 6-phosphate, and fructose l&diphosphate were obtained from Calbiochem; ATP and creatme phosphate were Sigma products; other chemicals were Baker analyzed reagents. Reagents. These were much the same as described by Michelsen (10) 65
66
PARVIN
AND
SMITH
and were prepared as follows: (a) 10N hydrochloric acid. (5) 0.234% ammonium metavanadate-2.34 gm NI$VO, was dissolved in 500 ml of hot water, 28 ml of concentrated HCl was added, and the solution was made to 1 liter with distilled water after cooling. (c) 3.53% ammonium molybdate35.3 gm of (NH,)M0,0244HZ0 was dissolved in warm water and diluted to 1 liter. Ammonium nsetavccnudate-mol$date reagent (reagent I). To about 150 to 300 ml water, 14 ml of (a) and 10 ml of reagent (5) were added and mixed; finally 20 ml of reagent (c) was added and the volume made up to 1 liter with distilled water. PROCEDURE Samples containing from 10 to 150 meoles of orthophosphate are diluted to 1.5 ml with water in 14 X 125 mm test tubes and 3.0 ml of n-butanol is added to all the tubes. This is followed by addition of 1.5 ml of ammonium metavanadate-molybdate (reagent I) and the tube is immediately shaken for 6 to 10 set either by hand or on a Vortex mixer. The absorbancy of the clear butanol layer obtained after standing for a few minutes .or ,after brief centrifugation is measured at 310 my against a reagent. blank. If the butanol layer is turbid it is clarified by the addition of 0.05 ml of methanol before recording the absorbancy. As long as the relative volumes of the orthophosphate solution, butanol, and reagent I are kept in the ratio of 1: 2 : 1, any suitable volume may be chosen. RESULTS In several experimental procedures requiring the determination of orthophospbate in the presence of labile phosphates, the phosphomolybdate complex is usually extracted in a suitable organic solvent to. avoid the error ,due td molybdate-catalyzed acid hydrolysis of labile phosphates (6). The possibility of employing solvent extraction of the molybdovanadophosphoric acid complex formed in Michelsen’s procedure (10) was explored. When n-butanol was used for the extraction of the molybdovanadophosphate complex, formed under the conditions of Michelsen (10) using reagent I of acid strength up to ‘0.08 N, extraction of the complex was incomplete. However, subsequent experiments showed that if the butanol used for the extraction of the molybdovanadophosphate complex was acidified (0.6 ml of 0.1 N HCl to 100 ml of butanol) the complex could be completely extracted. The need for acidification of the butanol prior to extraction of the complex was obviated if the ‘acid strength of reagent I was increased to at least 0.12 N. When the orthophosphate color was developed in this way, an identical extinction, coefficient for the pliosphomolybdovanadate complex was found in the
INORGANIC
PHOSPHATE
67
DETERMINATION
aqueous and in the butanol method. The absorption spectrum of the molybdovanadophosphoric acid complex in butanol showed (Fig. 1) a. peak at 310 my, which was slightly lower than the reported maximum absorption of 315 rnp in water (10). Since in the Michelsen method variation in the acid concentration affected the complex formation as well
1
290
3;0
330 WAVELENGTH
350 rnp
FIQ. 1. Absorption spectra of molybdovanadophosphoric in butanol. Details were aa described under “‘Procedure.” reagent blank was measured against butaaol. Curves 2 and dovanadophoaphate derived from 0.1 rmole Pi obtained measured against reagent blank and butanol, respectively.
370
acid and reagent blanks Curve 1, absorption of 3, absorbance of molybby the present method
as the blank absorption, the effect of varying acidities on the butanol extraction procedure was determined and the results are shown in Figure 2. As is evident from these results the final acid strength could, be varied between 0.06 and 0.15 N (with respect to the aqueous phase) without appreciably affecting either the blank absorption or the color development in the butanol phase. Thus, unlike the method of Michelsen (lo),
68
PARVIN
AND
SMITH
the butanol extraction procedure allows a wider range of acidities. A standard plot for orthophosphate (Fig. 3) using the butanol extraction procedure showed that Beer’s law is followed for up to at least 166 mqoles of orthophosphate. The color in the butanol phase remained unchanged for up to 26 hr at room temperature. Phosphorus determination in the presence of labile phosphate esters. Samples of fructose 1,6-diphosphate, ATP, glucose l-phosphate, and 0.0
20.6-
X
0 Gi ta
yj 0.4
X
-
-x-
,/x-x-x-x
I
X
x-x-xwx ‘X
/
/
/
: E
0
WI zoz-
0.;2 FINAL
0.06 ACID
0.i NORMALITY
0.;4 f H20
Oh PHASE)
FIQ. 2. Effect of acid concentration on absorbance of reagent blank and phosphomolybdovanadate in butanol extraction procedure. To 0.1 smole Pr solution or water (for reagent blank) sufkient HCl was added to bring the final acidity to that indicated in the 3 ml aqueous layer. Volume was made up to 1.!5 ml with water; 3 ml butanol was added, followed by 1.5 ml of reagent I. Following extraction in butanol, phosphorue color (X) in butanol phase was measured at 310 rnp age&& corresponding reagent blank. Reagent blanks (0) were read against butanol shaken with equal volume of water.
creatine phosphate were analyzed for their orthophosphate content by the present method and compared with the results obtained by the method of Marsh (6). From the results presented in Table 1, it is seen that the orthophosphate contents of fructose 1,6-diphosphate, glucose l-phosphate, and ATP were similar as detected by the two methods, while for creatine phosphate the Marsh method gave nearly twice as much orthophosphate content as the present method. Since molybdate-catalyzed hydrolysis of certain labile phosphates is well known (4, 5), it appea,rs likely that the
INORGANIC
PHOSPHATE
DETERMINATION
Pi mp
FRI. 3. Standard
plot for orthophosphate.
moles
Details as described under “Procedure.”
higher molybdate concentration used in the Marsh method @S-fold that in the present method) was responsible for creatine phosphate hydrolysis. Although the Marsh method (6) makes use of citrate to chelate excess molybdate, the short contact of phosphate compounds with molybdate before citrate addition was sufhcient to hydrolyze very labile phosphates such as creatine phosphate (5). To check whether it was necessary to bind the excess molybdate in the present method, and to what extent the molybdate-catalyzed hydrolysis of labile phosphates in the aqueous phase contributes to error, several tubes containing phosphate compounds were subjected to the complete procedure except that the butanol layer remained unseparated from the aqueous phase for varying time intervals. When creatine phosphate was present, the increase in phosphate color in the butanol phase was 15-170/o in 1 hr. With glucose l-phosphate the color increase was never more than 1.5% in 1 hr. After separation of the aqueous and butanol layer, no further increase in the absorbance was seen in the butanol layer. Determination
TABLE 1 of Phosphate Contents of Labile Organic Phosphatea by the Marsh and bv the Present Method Molar
Labile
phosphates
Glucose l-phosphate Fructose 1,Bdiphoephate Creatine phosphate ATP
Present
per cent Pi content
method
2.81 1.40 3.25 4.9
&B obtained Marsh
by method
2.90 1.41 6.8 5.2
70
PARVIN
AND
SMITH
Interjering substances. Similar to other methods, the present procedure was also affected by the presence of higher amounts of citrate, ATP, and pyrophosphate. However, because of the marked sensitivity of the present procedure, it was possible to analyze the orthophosphate accurately in the presence of these inhibitory substances by taking smaller aliquots of samples representing no less than 0.01 mole of orthophosphate and no more than 1 pole of ADP, ATP, or citrate (0.5 eole in the case of pyrophosphate). Thus, orthophosphate could be determined by the present method despite a lOO-fold excess of citrate or ATP being present and interference is simply eliminated by taking smaller aliquots. The presence of small amounts of protein (up to 200 pg of rat liver supernatant protein or bovine serum albumin) did not interfere in the butanol extraction method provided the same amount of protein was present in the corresponding blank. At higher protein concentrations, part of the yellow colored complex remained absorbed on the precipitated protein and, therefore, protein precipitation prior to orthophosphate estimation was necessary. Trichloroacetic acid (up to 16 mg/ml) used for protein precipitation did not affect the final absorbance providing the blank received identical treatment. DISCUSSION The phosphomolybdovanadate complex formation method has been applied to total phosphorus determination in biological materials (11) as well as to the measurement of orthophosphate released during an adenosine triphosphatase reaction (12). In the latter case, due to simultaneous hydrolysis of ATP, it was required that absorbancies be measured at constant time intervals. The extraction of phosphomolybdovanadate in butanol under appropriate conditions markedly improved such a phosphorus estimation method. As shown by the present study, the interference due to the hydrolysis of labile phosphates was minimal compared to other procedures. Further, labile esters remain in contact with the acid phosphomolybdovanadate for only a short time. The color development is very rapid, requiring less than 2 min, and the complete extraction of the phosphomolybdovanadate complex in butanol occurs within a few seconds. Once color has been developed, subsequent contact between the butanol phase and the lower aqueous layer does not appreciably affect colors in the butanol phase when the labile phosphate compounds present are glucose l-phosphate, ATP, or fructose 1,8diphosphate. Molybdate-catalyzed acid hydrolysis of phosphate compounds appeared to have no effect in the butanol phase once the color had been developed and butanol extraction performed. Only when creatine phosphate, which is known to
INORGANIC
PHOSPHATE
DETERMINATION
71
be extremely acid-labile (5)) was present was it advantageous to separate the butanol layer immediately after color development. The sensitivity of the method and the stability of the color developed makes this procedure abundantly amenable to enzyme kinetic studies involving analysis of orthophosphate in the presence of labile phosphates. The stability of the colors in the presence of labile phosphates probably results from the removal of excess molybdate from the aqueous phase by the butanol extraction. Furthermore, the initial molybdate concentration is very low. Vanadate, which is also present in low concentrations, does not accelerate acid hydrolysis of phosphate esters (5). In general, the present butanol extraction procedure offers distinct advantages over the Michelsen method since the present procedure tolerates much wider range of final acidities (between 0.06 to 0.15). Further, in the present procedure the blank absorptions are not affected by small variations in the acidity. In the procedure for phosphorus determination described in the experimental section of this paper, the final acidity is 0.07 N, which was appropriate for the estimation of phosphate in the presence of labile phosphates; however, when phosphate samples are free from labile phosphates, any acid normality between 0.06 to 0.15 N may be chosen. Thus for phosphate determination in acid-digested samples, the acid strength of reagent I can be lowered to 0.042 N and a suitable aliquot of the aciddigested samples taken to give final acid normalities from 0.06 to 0.15. The phosphomolybdate vanadate complex formation, unlike phosphomolybdate formation (14, 15)) has been found to be free from interferences due to silicate, arsenate, and iron (13). The presence of excessive amounts of nucleoside di- and triphosphates, pyrophosphate, and citrate, which inhibit color development in methods based on phosphomolybdate formation (16-l@, also interferes in the presently described method. However, sensitivity of the present method allows analyses to be performed by taking suitable aliquots so as to keep the concentration of color inhibitory substances low. During several enzyme studies involving phosphate determination, the present procedure has proved extremely valuable because of the sensitivity and rapidity of the method. Since the presence of a small amount of protein does not interfere, addition of reagent I serves to terminate the reactions as well as to develop simultaneously the color that conveniently allows a large number of analyses to be performed with a great precision. SUMMARY
An operationally simple and rapid procedure of orthophosphate determination, based on the formation of a phosphomolybdovanadate complex,
72
PAWIN
AND SMITH
followed by its subsequent extraction into butanol, is described. The procedure is particularly advantageous for estimation of orthophosphate in the presence of labile phosphates, and yields stable colors. Conditions affecting the reliability of this procedure are evaluated. ACKNOWLEDGMENTS This work was supported in part by a grant from the United Statea Public Health Service (GM 13407) and by the Univemity of California Cancer Coordinating Committee. REFERENCES 1. FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 66, 375 (1925). 2. BERENBLUISS, I., AND CHAIN, E., Biochem. J. 32, 295 (19338). 3. MARTIN, J. B., AND DOTY, D. M., Anal. Chem. 21, 965 (1949). 4. LOWRY, 0. H., AND LOPEZ, J. A., J. Biol. Chem. 162, 421 (1946). 5. WEIL-MALHERBE, H., AND GREEN, R. H., Biochem. J. 49, 286 (1951). 6. MAIWH, B. B., B&him. Biophys. Acta 32, 357 (1959). 7. IMSANDE, J., AND EPHRUSSI, B., Science 144, 854 (1964). 8. MISSON, G., Chem. Ztg. 32, 633 (1908). 9. QUINLAN, K. P., DE SESA, M. A., Anal. Chem. 27, 1626 (1955). 10. MICHELSEN, 0. B., Anal. Chem. 29, 69 (1957). 11. KOENIQ, R. A., AND JOHNSON, C. R., Znd. Eng. Chem., Anal. Ed. 14, 155 (1942). 12. LECOCQ, J., AND INEST, G., Anal. B&hem. 15, 160 (1966). 13. KITSON, R. E., AND MELLON, M. G., Znd. Eng. Chem., Anal. Ed. 16, 379 (1944). 14. BOLTZ, D. F., AND MELLON, M. G., Anal. Chem. 19, 873 (1947). 15. WADELIN, C., AND MELLON, M. G., Anal. Chem. 25, 1668 (1953). 16. BLUM, J. J., AND CHAMBERS, R. W., Biochim. Biophys. Acta 18, 601 (1955). 17. R~R, G. W., J. Biol. Chsm. 230, 643 (1958). 18. D’ADAMO, JR., A. F., AND BROICH, J. R., Anal. Biochem. 4, 420 (1962).