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Determination of phosphate: Study of labile organic phosphate interference
CLINICA CHIMICA ACTA
DETERMINATION PHOSPHATE
E. S. BAGINSKI,
OF PHOSPHATE:
STUDY
OF LABILE
ORGANIC
INTERFERENCE*
P. P. FOB AND B. ZAK
Divisions of’ Rrsearck am2 Laboratories, Sirtai Hospital, the Lkpavtments of Pathology, Physiology alzd Pharmacology, Wa_yne State Unioersity School oj Medicine and Detroit Gewral Hospital, Detroit, Mich. (t?.S..S.) (Received July 14th. 1~66)
SUMMARY
4n investigation was carried out on a newer method for the determination of inorganic phosphate which is applicable to the study of phosphate-splitting enzymes. It offers a unique advantage: after the color reagent has been added any inorganic phosphate formed cannot react with molybdenum, for the latter has been complexed by the addition of a citrate-arsenite solution and phosphate cannot compete successfully for the molybdate. This procedure appears to be quite suitable for the determination of inorganic phosphate, phospholipids, nucleotides or enzyme-hy~lrolyzed phosphate.
The determination of inorganic phosphate in biological materials is often carried out in the presence of organic compounds containing labile phosphate. The liberation of phosphate from these compounds during the analysis can lead to errors. This interference is commonly encountered in enzymatic procedures where phosphate liberated from a substrate is the measure of enzyme activity. Any non-enzymatic hydrolysis of the substrate would contribute to the over-all phosphate concentration and erroneously indicate higher than actual enzyme activity. Hydrolysis of this type is usually caused by protein-precipitating strong acids employed for termination of enzymatic reactions. A suitable control run simultaneously with the sample corrects at least in part for the resulting error. However, the hydrolysis is continuous and often erratic making precise determination of phosphate very difficult. This paper discusses a simple and sensitive procedure for phosphate determination which eliminates error due to non-enzymatic hydrolysis of phosphate. A unique feature of the method concerns the fact that the reagents form a stable colored complex with the inorganic phosphate present, and any additional phosphate liberated after the reagents have been added cannot react. * Aided by a grant from the Detroit General Hospital Research Corporation and by grant AM06034, National Institutes of Arthritis and Metabolic Diseases, U.S. Public Health Service. Cl&z. China.
Acta, 15 (1967) x55-158
BAGINSKI
156
et al.
REAGENTS I. The substrates used were adenosine f-triphosphate (ATP) and inorganic pyrophosfihate (PPi). ATP was chosen because it is a common substrate for various enzymatic analyses. PPi was chosen because it is a substrate for inorganic pyrophosphatase and has recently attracted considerable attention in view of its possible relationship to glucose 6-phosphatase and pyrophosphate phosphotransferase l-3. 2. 2% ascorbic acid (Merck medium crystals) in 10% trichloroacetic acid (A-TCA). This reagent is stable for at least two weeks at room temperature, if stored in an amber glass bottle. Its stability can be prolonged to four weeks if nitrogen gas is allowed to bubble through it for several minutes at the time of preparation. The stability is also increased by the addition of IOO mg of EDTA per IOO ml of solution. 3. I% ammonium molybdate tetrahydrate in water (A-M). 4. 2% sodium citrate &hydrate and 2”/0 anhydrous sodium arsenite in 2:/o acetic acid (A-C). The method for phosphate determination was described previously4>5. For the purpose of this work it was modified by combining ascorbic acid and trichloroacetic acid into one solution. RESULTS
An experiment was devised to prove that if one adds more inorganic phosphate immediately following the addition of the color reagents no further formation of the
-
’
ADDED
-
5
’
P, 0.1 ml
Fig. I. Effect of addition of phosphate on color formation. Curve at left: plateau formation of stable color. Curve at right: similar color formation though phosphate addition followed color reagent addition. Fig. 2. Kate of hydrolysis of several concentrations of ATP in acid solution. represent stability of color formed after color reagents are added to aliquots intervals. Gin.
Chim. Acta,
15 (1967) 155-158
Horizontal at various
lines time
DETERMINATION OF PHOSPHATE
I57
reduced molybdenum color will result. Fig. I shows the development of absorbance for 2 phosphate solutions. The curve on the left side represents the data obtained from a solution containing the following reagents added in sequence: 1.om1 of A-TCA, 0.x ml of 1.5 mM phosphate solution, 0.5 ml of A-M, 1.0 ml of A-C and, at the time indicated by the arrow, 0.1 ml of water. The curve on the right side represents data obtained in the same manner, except that the 0.1 ml of water was replaced by an additional 0.1 ml of 1.5 mM phosphate solution, as indicated by the arrow, immediately after the addition of the A-C reagent near zero time. It is apparent from the graph that the experiment was successful for once the A-C reagent had been added and the excess molybdate complexed the additional 0.1 ml of 1.5 m.iW of phosphate had no effect on the formation of color which remained stable during the several hours of the experiment . In order to evaluate the effect of non-enzymatic hydrolysis, experiments were carried out using ATP and PPi. Fig. 2 illustrates the rate of hydrolysis of ATP by A-TCA expressed in micromoles of inorganic phosphate formed. The procedure was the same as the one described above, except that the inorganic phosphate solution was replaced by 0.1 ml of 0.1 M, 0.~5 M and 0.2 M solutions of ATP respectively. The abcissa represents the time interval between the mixing of ATP with A-TCA and the addition of A-M. The addition of A-C followed immediately thereafter. It will be noted that after the addition of A-C reagent the color intensities shown as plateaus of absorbance (horizontal lines) stayed constant. Fig. z also indicates that the rate of hydrolysis of ATP appears to decrease as the concentration increases owing to binding of molybdate when ATP is in very high concentration. Fig. 3 shows the results of a similar experiment on the hydrolysis of PPi. In this experiment, performed as described above, 0.1 ml of 32 mM sodium pyrophosphate in 0.4 M sodium acetate buffer at pH 5.4 was used instead of the phosphate or the ATP solutions. The buffer was added to simulate the conditions under which
Fig. 3. Rate of hydrolysis of PPi in acid solution showing plateaus of stable colors after color reagents are added to aliquots at various time intervals. Clin. China. Acta, 15 (1967) 155-158
BAGINSKI
15s
et al.
this enzymatic hydrolysis is generally studied6. The absorbance plateaus again indicate that the continued hydrolysis of the substrate did not cause an increase in the molybdenum blue color even though hydrolysis under the acid circumstances of the experiment must still be occurring at the rate indicated by the ascending curve of Fig. 3. Though not shown in Fig. 3, the rate of hydrolysis of PPi varied inversely with its concentration as was the case with ATP. Figs. 2 and 3 indicate that non-enzymatic hydrolysis taking place during the short period following the addition of A-TCA and before the addition of the reagents is insignificant. Any hydrolysis occurring after the addition of the reagents does not interfere with the results. In working with enzyme suspensions or purified preparation of low protein content where no visible turbidity is formed upon the addition of A-TCA, the centrifugation step can be omitted and the color reagents can be added immediately. DISCUSSION
The sequence in which the reagents are added is critical for the success of the method. This suggests the following mechanism of reaction : inorganic phosphate combines instantaneously with molybdate to form an activated complex which in turn is reduced by ascorbic acid to form a color, which develops over a period of 15 min. The A-C reagent complexes the excess molybdate making it unavailable for reaction with any phosphate liberated after that moment. In addition, if the A-C reagent is not added some unfavorable side reactions come into play. Instead of a clear blue solution, a slightly opalescent greenish-blue color forms erratically and increases the absorbance of the reaction mixture out of proportion to the phosphate concentration. The amount of the reduced molybdenum complex formed appears to be dependent on the phosphate concentration, the nature of the reducing agent and the acidity of the final mixture7y8. When arsenite and citrate are present the reaction generates a definite amount of color concentration linearly related to the amount of phosphate present. The activation of molybdenum by phosphate has long been known to be due to the formation of a “twelve” acid, H,PO, . IO MOO, . Mo,O, (ref. 9). It is this compound of the many possible compounds of molybdenum which is easily reduced to the molybdenum blue state. For each phosphate concentration, once a fixed amount of molybdenum is reduced, the reaction ceases. It may very well be that the product of the reaction inhibits the formation of anv further color, for the final color is stable during a 24-h waiting period. REFEKENCES I 2 3 4 5 b 7 8
G. R. M. E. E. M. F. F.
W. RAFTER, 1. Bid. Chem.. 2x5 (1960) 1475. C. NORDLIE &D W. J. ARION,-J’. sioi. C&&, 239 (1964) 1680. R. STETTEN AND H. L. TAFT, I. Biol. Chem., 2x9 (1964) 4041. BAGINSICI AND B. ZAK, Clin. C&m. Acta, 5 (1960) i$+. BAGINSKI, L. WEINER AND B. ZAK, Clin. Chim. Acta, IO (1964) 378. K. STETTEN, J. Biol. Chem., 239 (1964) 3576. FEIGL, Spot Tests in Inovganic Analvsis, 5th ed., Elsevier, Amsterdam/New York, 1958. FEIGL, Chemistry of Specific, Select&e and Sensitive Reactions, Academic Press, New York,
1949. g P. KRUNHOLZ, Z. Anovg. Clin. Chim.
Acta,
Allgem.
15 (1967) 155-158
Chem..
21% (1~33) 97.
DETERMINATION PHOSPHATE
E. S. BAGINSKI,
OF PHOSPHATE:
STUDY
OF LABILE
ORGANIC
INTERFERENCE*
P. P. FOB AND B. ZAK
Divisions of’ Rrsearck am2 Laboratories, Sirtai Hospital, the Lkpavtments of Pathology, Physiology alzd Pharmacology, Wa_yne State Unioersity School oj Medicine and Detroit Gewral Hospital, Detroit, Mich. (t?.S..S.) (Received July 14th. 1~66)
SUMMARY
4n investigation was carried out on a newer method for the determination of inorganic phosphate which is applicable to the study of phosphate-splitting enzymes. It offers a unique advantage: after the color reagent has been added any inorganic phosphate formed cannot react with molybdenum, for the latter has been complexed by the addition of a citrate-arsenite solution and phosphate cannot compete successfully for the molybdate. This procedure appears to be quite suitable for the determination of inorganic phosphate, phospholipids, nucleotides or enzyme-hy~lrolyzed phosphate.
The determination of inorganic phosphate in biological materials is often carried out in the presence of organic compounds containing labile phosphate. The liberation of phosphate from these compounds during the analysis can lead to errors. This interference is commonly encountered in enzymatic procedures where phosphate liberated from a substrate is the measure of enzyme activity. Any non-enzymatic hydrolysis of the substrate would contribute to the over-all phosphate concentration and erroneously indicate higher than actual enzyme activity. Hydrolysis of this type is usually caused by protein-precipitating strong acids employed for termination of enzymatic reactions. A suitable control run simultaneously with the sample corrects at least in part for the resulting error. However, the hydrolysis is continuous and often erratic making precise determination of phosphate very difficult. This paper discusses a simple and sensitive procedure for phosphate determination which eliminates error due to non-enzymatic hydrolysis of phosphate. A unique feature of the method concerns the fact that the reagents form a stable colored complex with the inorganic phosphate present, and any additional phosphate liberated after the reagents have been added cannot react. * Aided by a grant from the Detroit General Hospital Research Corporation and by grant AM06034, National Institutes of Arthritis and Metabolic Diseases, U.S. Public Health Service. Cl&z. China.
Acta, 15 (1967) x55-158
BAGINSKI
156
et al.
REAGENTS I. The substrates used were adenosine f-triphosphate (ATP) and inorganic pyrophosfihate (PPi). ATP was chosen because it is a common substrate for various enzymatic analyses. PPi was chosen because it is a substrate for inorganic pyrophosphatase and has recently attracted considerable attention in view of its possible relationship to glucose 6-phosphatase and pyrophosphate phosphotransferase l-3. 2. 2% ascorbic acid (Merck medium crystals) in 10% trichloroacetic acid (A-TCA). This reagent is stable for at least two weeks at room temperature, if stored in an amber glass bottle. Its stability can be prolonged to four weeks if nitrogen gas is allowed to bubble through it for several minutes at the time of preparation. The stability is also increased by the addition of IOO mg of EDTA per IOO ml of solution. 3. I% ammonium molybdate tetrahydrate in water (A-M). 4. 2% sodium citrate &hydrate and 2”/0 anhydrous sodium arsenite in 2:/o acetic acid (A-C). The method for phosphate determination was described previously4>5. For the purpose of this work it was modified by combining ascorbic acid and trichloroacetic acid into one solution. RESULTS
An experiment was devised to prove that if one adds more inorganic phosphate immediately following the addition of the color reagents no further formation of the
-
’
ADDED
-
5
’
P, 0.1 ml
Fig. I. Effect of addition of phosphate on color formation. Curve at left: plateau formation of stable color. Curve at right: similar color formation though phosphate addition followed color reagent addition. Fig. 2. Kate of hydrolysis of several concentrations of ATP in acid solution. represent stability of color formed after color reagents are added to aliquots intervals. Gin.
Chim. Acta,
15 (1967) 155-158
Horizontal at various
lines time
DETERMINATION OF PHOSPHATE
I57
reduced molybdenum color will result. Fig. I shows the development of absorbance for 2 phosphate solutions. The curve on the left side represents the data obtained from a solution containing the following reagents added in sequence: 1.om1 of A-TCA, 0.x ml of 1.5 mM phosphate solution, 0.5 ml of A-M, 1.0 ml of A-C and, at the time indicated by the arrow, 0.1 ml of water. The curve on the right side represents data obtained in the same manner, except that the 0.1 ml of water was replaced by an additional 0.1 ml of 1.5 mM phosphate solution, as indicated by the arrow, immediately after the addition of the A-C reagent near zero time. It is apparent from the graph that the experiment was successful for once the A-C reagent had been added and the excess molybdate complexed the additional 0.1 ml of 1.5 m.iW of phosphate had no effect on the formation of color which remained stable during the several hours of the experiment . In order to evaluate the effect of non-enzymatic hydrolysis, experiments were carried out using ATP and PPi. Fig. 2 illustrates the rate of hydrolysis of ATP by A-TCA expressed in micromoles of inorganic phosphate formed. The procedure was the same as the one described above, except that the inorganic phosphate solution was replaced by 0.1 ml of 0.1 M, 0.~5 M and 0.2 M solutions of ATP respectively. The abcissa represents the time interval between the mixing of ATP with A-TCA and the addition of A-M. The addition of A-C followed immediately thereafter. It will be noted that after the addition of A-C reagent the color intensities shown as plateaus of absorbance (horizontal lines) stayed constant. Fig. z also indicates that the rate of hydrolysis of ATP appears to decrease as the concentration increases owing to binding of molybdate when ATP is in very high concentration. Fig. 3 shows the results of a similar experiment on the hydrolysis of PPi. In this experiment, performed as described above, 0.1 ml of 32 mM sodium pyrophosphate in 0.4 M sodium acetate buffer at pH 5.4 was used instead of the phosphate or the ATP solutions. The buffer was added to simulate the conditions under which
Fig. 3. Rate of hydrolysis of PPi in acid solution showing plateaus of stable colors after color reagents are added to aliquots at various time intervals. Clin. China. Acta, 15 (1967) 155-158
BAGINSKI
15s
et al.
this enzymatic hydrolysis is generally studied6. The absorbance plateaus again indicate that the continued hydrolysis of the substrate did not cause an increase in the molybdenum blue color even though hydrolysis under the acid circumstances of the experiment must still be occurring at the rate indicated by the ascending curve of Fig. 3. Though not shown in Fig. 3, the rate of hydrolysis of PPi varied inversely with its concentration as was the case with ATP. Figs. 2 and 3 indicate that non-enzymatic hydrolysis taking place during the short period following the addition of A-TCA and before the addition of the reagents is insignificant. Any hydrolysis occurring after the addition of the reagents does not interfere with the results. In working with enzyme suspensions or purified preparation of low protein content where no visible turbidity is formed upon the addition of A-TCA, the centrifugation step can be omitted and the color reagents can be added immediately. DISCUSSION
The sequence in which the reagents are added is critical for the success of the method. This suggests the following mechanism of reaction : inorganic phosphate combines instantaneously with molybdate to form an activated complex which in turn is reduced by ascorbic acid to form a color, which develops over a period of 15 min. The A-C reagent complexes the excess molybdate making it unavailable for reaction with any phosphate liberated after that moment. In addition, if the A-C reagent is not added some unfavorable side reactions come into play. Instead of a clear blue solution, a slightly opalescent greenish-blue color forms erratically and increases the absorbance of the reaction mixture out of proportion to the phosphate concentration. The amount of the reduced molybdenum complex formed appears to be dependent on the phosphate concentration, the nature of the reducing agent and the acidity of the final mixture7y8. When arsenite and citrate are present the reaction generates a definite amount of color concentration linearly related to the amount of phosphate present. The activation of molybdenum by phosphate has long been known to be due to the formation of a “twelve” acid, H,PO, . IO MOO, . Mo,O, (ref. 9). It is this compound of the many possible compounds of molybdenum which is easily reduced to the molybdenum blue state. For each phosphate concentration, once a fixed amount of molybdenum is reduced, the reaction ceases. It may very well be that the product of the reaction inhibits the formation of anv further color, for the final color is stable during a 24-h waiting period. REFEKENCES I 2 3 4 5 b 7 8
G. R. M. E. E. M. F. F.
W. RAFTER, 1. Bid. Chem.. 2x5 (1960) 1475. C. NORDLIE &D W. J. ARION,-J’. sioi. C&&, 239 (1964) 1680. R. STETTEN AND H. L. TAFT, I. Biol. Chem., 2x9 (1964) 4041. BAGINSICI AND B. ZAK, Clin. C&m. Acta, 5 (1960) i$+. BAGINSKI, L. WEINER AND B. ZAK, Clin. Chim. Acta, IO (1964) 378. K. STETTEN, J. Biol. Chem., 239 (1964) 3576. FEIGL, Spot Tests in Inovganic Analvsis, 5th ed., Elsevier, Amsterdam/New York, 1958. FEIGL, Chemistry of Specific, Select&e and Sensitive Reactions, Academic Press, New York,
1949. g P. KRUNHOLZ, Z. Anovg. Clin. Chim.
Acta,
Allgem.
15 (1967) 155-158
Chem..
21% (1~33) 97.