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Colorimetric determination of orthophosphate in the assay of inorganic pyrophosphatase activity
ANALYTICAL
BIOCHEMISTRY
Calorimetric
49,
37-47
(1972)
Determination of
Inorganic
of Orthophosphate Pyrophosphatase
MOGENS University
Department
of Clinical 8000 Arhus Received
in the Assay Activity
H@RDER Chemistry, C, Denmark
November
Arhus
Kommunehospital,
3, 1971
The activity of inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) in biological material can be determined by following the enzyme-catalyzed change in the amount of the substrate, inorganic pyrophosphate, or the product, inorganic orthophosphate. Direct calorimetric determination in an unnecessarily difficult and timeconsuming analysis (1). Pyrophosphate may also be determined as orthophosphate after total hydrolysis, but this requires quantitative separation of the product, orthophosphate, and residual substrate, pyrophosphate, prior to hydrolysis (2). The use of P32-marked pyrophosphate as substrate (3,4) likewise requires quantit.ative separation of pyrophosphkte and orthophosphate, whether it is the change in the substrate or the change in the product which is determined. Direct measurement of the increase in the product, orthophosphate, is most frequently used without preceding removal of the remaining substrate, using Fiske and SubbaRow’s technique (5) : after the formation of the heteropoly acicl 12-molybdophosphoric acid in a strongly acidic solution, this compound is reduced to a mixture of complexes of hexavalent and pentavalent molybdenum with orthophosphate, called molybdenum blue. It is, however, an important problem that several phosphate compounds, including inorganic pyrophosphate, are hydrolyzed as a result of the high hydrogen ion concentration in the reagents. Baginski, Foa, and Zak (6) showed that adding arsenite-citrate immediately after the formation of the orthophosphate-molybdenum complexes prevented the inclusion of orthophosphate released later, because surplus molybdenum is irreversibly complex by arsenite-citrate. For this reason Hruska (7) used this method to determine the activity of inorganic pyrophosphatase on biological fluids. He found, however, that protein precipitation in the presence of 0.6 N trichloroacetic acid at room temperature resulted in approximately 2.5% of 5 pmoles pyrophosphate being changed to orthophosphate in 37 @ 1972 by Academic Press, Inc. All rights of reproduction in any
form
reserved.
38
MOGENS
HrdRDER
the course of 15 min. Weil-Malherbe and Green (8) have shown that molybdate caa catalyze hydrolysis of inorganic pyrophosphate and, finally, it is known (7,9) that pyrophosphate can interfere with the complex formation of orthophosphate-molybdate. There would thus seem to be several reasons for the reduction in the accuracy of measuring pyrophosphatase, both before and during the determination of orthophosphate. Inorganic pyrophosphatase activity is of importance in relation to the biological function of the nonspecific alkaline phosphomonoesterase (10) and in order to further explain the metabolism of the large amounts of inorganic pyrophosphate formed by the organism (11). This paper presents a method for determining inorganic pyrophosphatase activity in biological fluids, using Baginski and Zak’s (12) technique for determining orthophosphate as a basis, and evaluates a series of possible sources of error that may influence the accuracy and precision of the enzyme assay. METHOD
The total reaction volume for enzymic hydrolysis is 1 ml in the originally described procedure (12). Enzyme catalysis and protein precipitation is completed by adding 2 ml of 1.2 N trichloroacetic acid. Orthophosphate is then determined using 1 ml of the supernatant by adding 0.2 ml of 10% ascorbic acid, 0.5 ml of 1% ammonium molybdate, and 1.0 ml of 2% sodium arsenite-sodium citrate. Absorbance is then read in 10 mm cuvets at 700 nm. All chemicals are reagent grade, the water is double distilled, and all glassware is washed out in acid. As mentioned in the introduction the presence of inorganic pyrophosphate necessitates changes in the procedures for protein precipitation and estimation of inorganic orthophosphate. The basis for these changes and the method resulting from them is described in the following. RESULTS
Stoppage of Enzyme-Catalyzed
Hydrolysis
and Protein Precipitation
Approximately 0.15% of 3 pmoles inorganic pyrophosphate was hydrolyzed per hour in 0.6 N trichloroacetic acid at O”C, while stable at pH 4.0 (Fig. 1). As protein precipitation is necessary, trichloroacetic acid is required. For this reason the pyrophosphohydrolytic activity and protein precipitation ability of trichloroacetic acid were investigated in relation to the time of the reaction and temperature in 3 ml solutions containing 0.8 pmole pyrophosphate, 0.4 N trichloroacetic acid, and 0.1 ml of serum. The solutions were left for 5 and 30 min at a temperature of 0°C and at room temperature. Then the precipitate was sedimented
INORGANIC
PYROPHOSPHATASE
39
ACTIVITY
nmol LOr
0
Y
60
120
180
240
m’n.
FIG. 1. Nonenzymic hydrolysis of inorganic pyrophosphate at 0°C. 3 pmolcs pyrophosphate was dissolved in 0.6 N trichloroacetic acid (X) and in acetic acidacetate buffer, pH 4.0 (0). Orthophosphate release (ordinate) was determined and followed for 210 min.
by centrifugation and orthophosphate was determined in the supernatant (Table 1). It appears that the acid-catalyzed hydrolysis was, as expected, greater at room temperature than at 0°C. The uniform analysis dispersion shows that protein precipitation is also effective at 0” and seems to be completed after only 5 min. Catalysis at 0” was negligible after 30 min. Adspyrophosphate is stable at pH 4.0 (Fig. 1) and also at room temperature, the pH in the supernatant must be altered to this level. On account of the analysis process it is desirable to add constant buffer volume to supernatant. In a series of experiments, 1 ml of acetic acid-acetate buffer, concentrations 1.0-2.0 moles/liter and pH 4.5-5.0, was added to 2 ml of supernatant; 1 ml of acetic acid-acetate buffer, pH 5.0, 2.0 moles/liter proved to be suitable, giving a pH in mixture of 4.15. Hydrolysis of Inorganic Incubation t’ime, min Incubation t,emp., “C Mean release of orthophosphate S.1). (nmole)
TABLE; 1 Pyrophosphate
(nmole)
5 0” z 7 0.04
by Trichloroacetic :;o 0” 2. 1 0. Ott
Acid :i0 L”Ja 12.2 0. 16
Incubation mixture consisted of 0.8 rmole inorganic pyrophosphate, 0.3 mg human serum protein, and 0.4 N trichloroacetic acid. Eight estimations for each c:ondit,iorl.
Concentrations of Reagents in Orthophosphate Detel-mination Orthophosphate determination was carried out on a solution containing 0.04 prnole orthophosphate and 0.5 Ilmole pyrophosphate such that the concentrations of the reagents were varied one by one. The final
40
MOGENS
HGRDER
concentrations for the nonvarying reagents were those named under “Method,” ascorbic acid 0.720/o, ammonium molybdate 0.18% and arsenite-citrate 0.72%. The range in concentration which gives least change in absorbance by varying the concentration of the reagents were (Fig. 2) for ascorbic acid from approximately 0.7% to approximately 1.2%, for ammonium molybdate more than 0.40/o, and for arsenite-citrate more than 0.35%.
t
. 0.2
0.4
. 0.6
. OS
10
1.2
Y.
FIG. 2. Effect on final absorbances in orthophosphate assay of varying reagents concentrations one by one : ascorbic acid (X), ammonium molybdate (a), and arsenite-citrate (0). Final concentrations of reagents (abscissa) are given in per cent. Analyses were carried out on samples containing 0.004 pmole orthophosphate and 0.5 pmole inorganic pyrophosphate. Values on ordinate are absorbances of samples minus absorbances of reagent blanks.
“Timing”
of Orthophosphate Analysis
The time intervals between the addition of ascorbic acid and ammonium molybdate were tested on solutions containing 1.0 pmole pyrophosphate and orthophosphate from 0 to 0.16 pmole. Identical absorbance values were found for reagent blank, sample, and analytical dispersion, whether the reagents were added at an interval of 5 min or as a mixture. On the other hand the time lapse between the addition of ammonium molybdate and arsenite-citrate influenced the final absorbance values of a solution containing 1.0 pmole inorganic pyrophosphate (Fig. 3). The lowest value was found when arsenite-citrate was added immediately after ammonium molybdate: the immediate absorbance value in this case was equal to the final value. With time intervals of 10 and 20 min for adding reagents, the final absorbance values increased in direct proportion and furthermore 5-7 min elapsed before the immediate absorbance values had fallen to their final level. If arsenite-citrate was not added at all, there was a steadily increasing absorbance value. A molybdate-catalyzed hydrolysis of pyrophosphate would thus seem to
INORGANIC
PYROPHOSPHATASE
ACTIVITY
41
Abs
I 10
0
20
30
* min LO
FIG. 3. Effect on final absorbances in orthophosphate assay of varying the lag between addition of ammonium molybdate and arsenite-citrate reagents. time zero (a), 10 min ( x ), 20 min (0). Arsenite-citrate not added at all Abscissa: time after addition of ammonium molybdate.
time Lag (A).
occur. If orthophosphate is also present in the sample, the course of the development of absorbance is identical with that of the pure pyrophosphate solution. Absorbance falls by approximately O.S%/min in a period of 5 to 40 min after addition of arsenite-citrate. It is therefore necessary to fix the times for the addition of arsenite-citrate and the reading of the samples. Absorbance
Spectra
and Standard
Curves
Absorbance spectra for solutions containing (1) orthophosphate 0.08 pmole, (2) orthophosphate 0.04 pmole and pyrophosphate 2.00 ,umoles, and (3) pyrophosphate 2.00 pmoles all showed a maximum at approximately 700 nm and 850 nm (Fig. 4). There was linearity between absorbance and amount of orthophosphate up to 0.16 /Amole at both 713 Abs
Bkc. 4. Absorbance spectra for samples containing O.Og pmole orthophosphate (O), 0.04 @mole orthophosphate plus 2.00 ,umoles pyrophosphate (a) and 2.00 pmoles pyrophosphate (X). All readings were made in 10 mm cuvets against distilled water on a Zeiss PM& II spectrophotometer. Abscissa: wavelength values in nm. Ordinate: absorbance (sample - reagent blank).
42
MOGEKS
H@RDER
Abs.
0.04
0.06
0.12
016 rmol
FIG. 5. Orthophosphate standard curves at wavelengths 713 nm (0) and 850 nm orthophosphate in sample. Ordinate : absorbance (0). Abscissa : inorganic (sample - reagent blank).
nm and 850 nm (Fig. 5). Pyrophosphate in samples gave rise to identical absolute increase in absorbance for all amounts of orthophosphate, 0.080 and 0.120 absolute absorbance units at 713 nm and 850 nm, respectively, for 2.0 pmoles of pyrophosphate. Interference
In a solution containing 0.08 /*mole orthophosphate, absorbance increased in direct proportion to the increasing pyrophosphate concentration until approximately 2.0 mmoles/liter, afterwards falling so that when pyrophosphate concentration was 15 mmolesJliter orthophosphate Abs.
FIG. 6. Interference of inorganic pyrophosphate on orthophosphate assay on samples containing 0.08 ,umole orthophosphate and O-20 amoles inorganic pyrophosphate (0). Pyrophosphate blank samples as references (X). Abscissa: inorganic pyrophosphate in sample.
INORGANIC
PYROPHOSPHATASE
ACTIVITY
43
FIG. 7. Recovery of 0.08 pmole orthophosphnte in samples containing inorganic pyrophosphate (X ), adenosindiphosphate ( q ) , P-glycelophosphate (O), and pof phosphate compounds nitrophenyl phosphate (8). Abscissa: concentrations other than orthophosphate.
could not be recovered after compensating for pyrophosphate blank value (Fig. 6). Other phosphate compounds which are also used as phosphohydrolass substrates interfere likewise with orthophosphate determination. Solutions with adenosine diphosphate, /?-glycerophosphate, and p-nitrophenyl phosphate in concentrations between 0.5 and 20 mmoles/liter caused, au did gyrophosphate, a decrease in the recovery of 0.08 blmole orthophosphate, but such that interference declines in the order mentioned (Fig. 7). In order to clarify further t,he process of pyrophosphatc interference, 10 ,umolespyrophosphate was added to a solution containing 0.06 qole orthophosphate at various times during the orthophosphate analysis procedure. According to the results in Fig. 7, a 10% recovery of orthophosphate could be expected. This was also found when pyrophosphate was added at any time before ammonium molybdate or together with it (Fig. 8). The longer the time lapse from the addition of ammonium molybdate, the less pyrophosphate interfered. Pyrophosphnte did not. interfere at all when added together with arsenite-citrate or later. As acetate buffer is used to lower the hydrogen ion concentration in the supernatant after protein precipitation, the recovery of 0.04 and 0.08 pmole orthophosphate in sodium acetate was investigated (Table 2). Recovery was 100% in the case of t,he acetate buffer concentration of 0.7 mole/liter used, but higher acetate concentrations caused interference. Some inorganic and organic compounds which are employed in the systematic investigation of enzyme activity interfered to varying degrees (Fig. 9). Magnesium sulfate, glucose, and urea did not interfere at all in the concentration range investigated up to 1.0 mole/liter. Borate inter-
44
MOGENS
I
. 0
5
H$RDER
IO
I5
20 min.
FIG. 8. Effect of adding, at different times during orthophosphate assay procedure, 10 amoles inorganic pyrophosphate (X) to samples containing 0.06 pmole orthophosphate. 109% reference value is a sample where no pyrophosphate is added at all. Abscissa: time in orthophosphate procedure. Ordinate: per cent recovery of orthophosphate.
TABLE 2 of Acetate on Orthophosphate
Interference
Assay
Orthophosphate in sample, mmole/liter
Sodium acetate in sample, mole/liter 0.05
0.25
0.50
1.00
1.50
2.00
2.50
0.04 0.08
95 98
99 102
100 100
97 98
91 92
79 77
73 72
Figures indicate percentage recovery of orthophosphate
0
0.1
0.2
0.3
01
in samples.
0.5 molll
FIG. 9. Recovery in per cent of 0.04 pmole orthophosphate in samples containing magnesium sulfate or urea or glucose (e), borate (X ), Tris (A), potassium iodide (‘O), sodium citrate (+ ), and lithium chloride or sodium chloride or potassium chloride (0). Abscissa: concentrations of interfering substances in sample.
INORGANIC
PYROPHOSPHATASE
45
ACTIVITY
fered slightly. Lithium, sodium, and potassium chlorides caused uniform interference with an orthophosphate recovery of approximately 15% at a chloride concentration of 0.4 mole/liter and over. Tris, potassium iodide, and sodium citrate inhibited almost totally with concentrations of 0.5, 0.1, and 0.1 mole/liter, respectively. Final Procedure When hydrolysis has ended, 1 ml of 1.2 N (20%) trichloroacetic acid at 0°C was added to 2 ml of enzyme reaction mixture. After standing for 10 min at O-4”, the mixture was centrifuged for 10 min at o-4”. Then 1 ml of acetic acid-sodium acetate buffer, 2.0 moles/liter, pH 5.0, was added to 2 ml of supernatant; 1 ml of this mixture was taken for orthophosphate analysis, double analysis. At a fixed time, 0.7 ml of 10% ascorbic acid and 3% ammonium molybdate was added, freshly mixed in the proportion 2 + 5; 10 min later 1 ml of 2% sodium arsenite-sodium citrate in 2% (v/v) acetic acid was added. The absorbance was read after 10 min at 700 or 850 nm. Orthophosphate amounts of 0.04 and 0.08 pmole dissolved in a reaction mixture without enzyme were taken as standards. At the same time possible interference will be disclosed. Reliability
of Determination
of Pyrophosphatase
Activity
The sensitivity of the pyrophosphatase assay is maximally that of the orthophosphate analysis. This is approximately 0.0025 pmole orthophosphate, as this amount corresponds to an apparatus reading of 0.010 absolute absorbance unit at 10 mm light path at, 713 nm. Given 2 ml of enzyme activity reaction mixture and a reaction time of 5 min, an enzyme-catalyzed hydrolysis of 0.0005 pmole pyrophosphate per minute can be shown. The accuracy of the enzymic pyrophosphate phosphohydrolysis itself has not been investigated, but is ensured partly by the elimination of the nonenzyme-catalyzed hydrolysis of pyrophosphate caused by trichloroacetic acid and molybdate, partly by the detection of, and compensation for interfcrcnce with the orthophosphate analysis which ensures 100% recovery of orthophosphate. The precision of the analysis is also partly determined by the precision of the orthophosphate analysis. The coefficient of variation in 43 analyses of aqueous orthophosphate solutions carried out over a period of 3 months was 2.5%. The minimum coefficient of variation was 0.7% in 10 repeated analyses all on the same day. The precision of pyrophosphatase determination was investigated by performing 10 consecutive activit,y determinations on the same enzyme solution. Average activity was 155 IUJliter (,umole pyrophosphate X min-l X 1 enzyme-l), standard deviation was 3 III/liter, and the coefficient of variation thus 2.07~. 33 sets
46
MOGENS
H@RDER
of double determinations of enzyme activity on different enzyme material with widely differing activity gave a dispersion of double determinations of the order of 0.0003 IU. DISCUSSION
The fact that enzyme activity is quantitatively expressed by the amount of converted substrate raises two analytical problems: (1) partly that substrate consumption during the whole analytical procedure should be a result of the enzyme’s catalytic activity only, and (2) partly that one has to be able to quantitate the amount of converted substrate accurately, either directly or as increase in product growth. The trichloroacetic acid catalysis accompanying protein precipitation which has been shown here is well known from work with acid-labile compounds (13). A high trichloroacetic acid concentration, temperature at approximately room temperature, and longer reaction time increase protein precipitation ability, but the same conditions increase pyrophosphate hydrolysis at the same time. The conditions chosen are therefore a compromise in which a minimal hydrolysis of pyrophosphate can still take place. The same is true of the proved molybdate-catalyzed hydrolysis of pyrophosphate. It is, however, of vital importance in choosing Baginski and Zak’s method (12) that any hydrolysis of pyrophosphate after the addition of arsenite-citrate is of no importance. The development of absorbance found in pyrophosphate solutions after the addition of arsenite-citrate at different times after molybdate could make one suppose that special pyrophosphate molybdate complexes are formed. This will influence the accuracy of the orthophosphatNe analysis. The uniform absorbance spectra for pyrophosphate and orthophosphate solutions are against this view, but the problem is not completely clear. Control of the possibility of interference which is usual for all methods based on molybdate complex formation (14) is of decisive importance for the accuracy of the orthophosphate analysis. Pyrophosphate interferes at such low concentrations that dilution is necessary when the concentration in the enzyme reaction mixture rises above 4.5 mmoles/ liter, equivalent to 2.0 mmoles/liter in the acetate buffer neutralized supernatant used for the orthophosphate analysis. Pyrophosphate seems to interfere with the formation of molybdate-orthophosphate complexes and so the process is similar to the generally known one (14). Interference due to frequently used buffer components and salts illustrates the necessity of controlling orthophosphate standards as “internal.” This means that standards must be included every time the reaction mixture’s composition is changed. In systematic investigations
INORGANIC
PYROPHOSPHATASE
ACTIVITY
47
of enzyme activity it is, however, especially necessary to be able to show whether buffer or salt effect is an actual fact or a simple analytical phenomenon having nothing to do with enzyme activity at all. In a future article, examples of salt and buffer effects of both types will be given, using the pyrophosphatase assay for determining the enzyme activity in human serum. SUMMARY
Using Baginski and Zak’s (12) method for determining inorganic orthophosphate as a starting point, a number of conditions which influence the accuracy and precision of the determination of pyrophosphatase activity have been shown: nonenzyme-catalyzed acid hydrolysis by protein precipitat,ion agents and molybdate-catalyzed hydrolysis of pyrophosphate together with interference in the determination of orthophosphate by the substrate pyrophosphate and other components from the reaction mixture, Tris, chloride, acetate, citrate, boric acid. With regard to these sources of error, a method is described for determining l)yrophosphatase activity, and its reliability is investigated. REFERENCES 1. FLYNN. R. M., JONES, M. E., .~ND LIPM.~IXN, F. A., J. BioE. Chem. 211, 791 (1954). 2. BAILEY, K., in “Methods of Enzymatic Analysis” (Bergmeyer, H.-U., ed.), pp. 644-647. Academic Press, New York/London, 1963. 3. BLOEMERS. H. P. J., STEPHENSON, M. L., AND ZAMECNII~, P. C., Anal. Biochern. 34, 66 (1970). 4. HEINONEN. J., Anal. Biochem. 37, 32 (1970). 5. FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 66, 375 (1925). 6. BAGINSKI, E. S., FOA, P. P., AND ZAK, B., Clin. Chim. Acta 15, 155 (1967). 7. H~us~ta, K. J., Cb. Chim. Acta 22, 454 (1968). S. WEIL-MALHERBE. H., AND GREEN. R. H., Biochem. J. 49, 286 (1951). 9. PARYIN, R., AND SMITH, R. A., Anal. Biochem. 27, 65 (1969). 10. POSEN, S., Ann. Int. Med. 67, 183 (1967). 11. KORNBERC. A.. in “Horizons in Biochemistry” (Kashn, A., and Pullman, B. cds.), pp. 251-264. Academic Press. New York/London, 1962. 12. BAGINSW E. S., AND ZAK, B., Clin. Chim. Actn 5, 834 (1960). 13. LOWRY, 0. H.. AND LOPEZ, J. A,. J. Biol. Chem. 162, 421 (1946). 14. BOLTZ. D. F.. AND MELLON, M. G., And. Chem. 19, 873 (1947).
BIOCHEMISTRY
Calorimetric
49,
37-47
(1972)
Determination of
Inorganic
of Orthophosphate Pyrophosphatase
MOGENS University
Department
of Clinical 8000 Arhus Received
in the Assay Activity
H@RDER Chemistry, C, Denmark
November
Arhus
Kommunehospital,
3, 1971
The activity of inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) in biological material can be determined by following the enzyme-catalyzed change in the amount of the substrate, inorganic pyrophosphate, or the product, inorganic orthophosphate. Direct calorimetric determination in an unnecessarily difficult and timeconsuming analysis (1). Pyrophosphate may also be determined as orthophosphate after total hydrolysis, but this requires quantitative separation of the product, orthophosphate, and residual substrate, pyrophosphate, prior to hydrolysis (2). The use of P32-marked pyrophosphate as substrate (3,4) likewise requires quantit.ative separation of pyrophosphkte and orthophosphate, whether it is the change in the substrate or the change in the product which is determined. Direct measurement of the increase in the product, orthophosphate, is most frequently used without preceding removal of the remaining substrate, using Fiske and SubbaRow’s technique (5) : after the formation of the heteropoly acicl 12-molybdophosphoric acid in a strongly acidic solution, this compound is reduced to a mixture of complexes of hexavalent and pentavalent molybdenum with orthophosphate, called molybdenum blue. It is, however, an important problem that several phosphate compounds, including inorganic pyrophosphate, are hydrolyzed as a result of the high hydrogen ion concentration in the reagents. Baginski, Foa, and Zak (6) showed that adding arsenite-citrate immediately after the formation of the orthophosphate-molybdenum complexes prevented the inclusion of orthophosphate released later, because surplus molybdenum is irreversibly complex by arsenite-citrate. For this reason Hruska (7) used this method to determine the activity of inorganic pyrophosphatase on biological fluids. He found, however, that protein precipitation in the presence of 0.6 N trichloroacetic acid at room temperature resulted in approximately 2.5% of 5 pmoles pyrophosphate being changed to orthophosphate in 37 @ 1972 by Academic Press, Inc. All rights of reproduction in any
form
reserved.
38
MOGENS
HrdRDER
the course of 15 min. Weil-Malherbe and Green (8) have shown that molybdate caa catalyze hydrolysis of inorganic pyrophosphate and, finally, it is known (7,9) that pyrophosphate can interfere with the complex formation of orthophosphate-molybdate. There would thus seem to be several reasons for the reduction in the accuracy of measuring pyrophosphatase, both before and during the determination of orthophosphate. Inorganic pyrophosphatase activity is of importance in relation to the biological function of the nonspecific alkaline phosphomonoesterase (10) and in order to further explain the metabolism of the large amounts of inorganic pyrophosphate formed by the organism (11). This paper presents a method for determining inorganic pyrophosphatase activity in biological fluids, using Baginski and Zak’s (12) technique for determining orthophosphate as a basis, and evaluates a series of possible sources of error that may influence the accuracy and precision of the enzyme assay. METHOD
The total reaction volume for enzymic hydrolysis is 1 ml in the originally described procedure (12). Enzyme catalysis and protein precipitation is completed by adding 2 ml of 1.2 N trichloroacetic acid. Orthophosphate is then determined using 1 ml of the supernatant by adding 0.2 ml of 10% ascorbic acid, 0.5 ml of 1% ammonium molybdate, and 1.0 ml of 2% sodium arsenite-sodium citrate. Absorbance is then read in 10 mm cuvets at 700 nm. All chemicals are reagent grade, the water is double distilled, and all glassware is washed out in acid. As mentioned in the introduction the presence of inorganic pyrophosphate necessitates changes in the procedures for protein precipitation and estimation of inorganic orthophosphate. The basis for these changes and the method resulting from them is described in the following. RESULTS
Stoppage of Enzyme-Catalyzed
Hydrolysis
and Protein Precipitation
Approximately 0.15% of 3 pmoles inorganic pyrophosphate was hydrolyzed per hour in 0.6 N trichloroacetic acid at O”C, while stable at pH 4.0 (Fig. 1). As protein precipitation is necessary, trichloroacetic acid is required. For this reason the pyrophosphohydrolytic activity and protein precipitation ability of trichloroacetic acid were investigated in relation to the time of the reaction and temperature in 3 ml solutions containing 0.8 pmole pyrophosphate, 0.4 N trichloroacetic acid, and 0.1 ml of serum. The solutions were left for 5 and 30 min at a temperature of 0°C and at room temperature. Then the precipitate was sedimented
INORGANIC
PYROPHOSPHATASE
39
ACTIVITY
nmol LOr
0
Y
60
120
180
240
m’n.
FIG. 1. Nonenzymic hydrolysis of inorganic pyrophosphate at 0°C. 3 pmolcs pyrophosphate was dissolved in 0.6 N trichloroacetic acid (X) and in acetic acidacetate buffer, pH 4.0 (0). Orthophosphate release (ordinate) was determined and followed for 210 min.
by centrifugation and orthophosphate was determined in the supernatant (Table 1). It appears that the acid-catalyzed hydrolysis was, as expected, greater at room temperature than at 0°C. The uniform analysis dispersion shows that protein precipitation is also effective at 0” and seems to be completed after only 5 min. Catalysis at 0” was negligible after 30 min. Adspyrophosphate is stable at pH 4.0 (Fig. 1) and also at room temperature, the pH in the supernatant must be altered to this level. On account of the analysis process it is desirable to add constant buffer volume to supernatant. In a series of experiments, 1 ml of acetic acid-acetate buffer, concentrations 1.0-2.0 moles/liter and pH 4.5-5.0, was added to 2 ml of supernatant; 1 ml of acetic acid-acetate buffer, pH 5.0, 2.0 moles/liter proved to be suitable, giving a pH in mixture of 4.15. Hydrolysis of Inorganic Incubation t’ime, min Incubation t,emp., “C Mean release of orthophosphate S.1). (nmole)
TABLE; 1 Pyrophosphate
(nmole)
5 0” z 7 0.04
by Trichloroacetic :;o 0” 2. 1 0. Ott
Acid :i0 L”Ja 12.2 0. 16
Incubation mixture consisted of 0.8 rmole inorganic pyrophosphate, 0.3 mg human serum protein, and 0.4 N trichloroacetic acid. Eight estimations for each c:ondit,iorl.
Concentrations of Reagents in Orthophosphate Detel-mination Orthophosphate determination was carried out on a solution containing 0.04 prnole orthophosphate and 0.5 Ilmole pyrophosphate such that the concentrations of the reagents were varied one by one. The final
40
MOGENS
HGRDER
concentrations for the nonvarying reagents were those named under “Method,” ascorbic acid 0.720/o, ammonium molybdate 0.18% and arsenite-citrate 0.72%. The range in concentration which gives least change in absorbance by varying the concentration of the reagents were (Fig. 2) for ascorbic acid from approximately 0.7% to approximately 1.2%, for ammonium molybdate more than 0.40/o, and for arsenite-citrate more than 0.35%.
t
. 0.2
0.4
. 0.6
. OS
10
1.2
Y.
FIG. 2. Effect on final absorbances in orthophosphate assay of varying reagents concentrations one by one : ascorbic acid (X), ammonium molybdate (a), and arsenite-citrate (0). Final concentrations of reagents (abscissa) are given in per cent. Analyses were carried out on samples containing 0.004 pmole orthophosphate and 0.5 pmole inorganic pyrophosphate. Values on ordinate are absorbances of samples minus absorbances of reagent blanks.
“Timing”
of Orthophosphate Analysis
The time intervals between the addition of ascorbic acid and ammonium molybdate were tested on solutions containing 1.0 pmole pyrophosphate and orthophosphate from 0 to 0.16 pmole. Identical absorbance values were found for reagent blank, sample, and analytical dispersion, whether the reagents were added at an interval of 5 min or as a mixture. On the other hand the time lapse between the addition of ammonium molybdate and arsenite-citrate influenced the final absorbance values of a solution containing 1.0 pmole inorganic pyrophosphate (Fig. 3). The lowest value was found when arsenite-citrate was added immediately after ammonium molybdate: the immediate absorbance value in this case was equal to the final value. With time intervals of 10 and 20 min for adding reagents, the final absorbance values increased in direct proportion and furthermore 5-7 min elapsed before the immediate absorbance values had fallen to their final level. If arsenite-citrate was not added at all, there was a steadily increasing absorbance value. A molybdate-catalyzed hydrolysis of pyrophosphate would thus seem to
INORGANIC
PYROPHOSPHATASE
ACTIVITY
41
Abs
I 10
0
20
30
* min LO
FIG. 3. Effect on final absorbances in orthophosphate assay of varying the lag between addition of ammonium molybdate and arsenite-citrate reagents. time zero (a), 10 min ( x ), 20 min (0). Arsenite-citrate not added at all Abscissa: time after addition of ammonium molybdate.
time Lag (A).
occur. If orthophosphate is also present in the sample, the course of the development of absorbance is identical with that of the pure pyrophosphate solution. Absorbance falls by approximately O.S%/min in a period of 5 to 40 min after addition of arsenite-citrate. It is therefore necessary to fix the times for the addition of arsenite-citrate and the reading of the samples. Absorbance
Spectra
and Standard
Curves
Absorbance spectra for solutions containing (1) orthophosphate 0.08 pmole, (2) orthophosphate 0.04 pmole and pyrophosphate 2.00 ,umoles, and (3) pyrophosphate 2.00 pmoles all showed a maximum at approximately 700 nm and 850 nm (Fig. 4). There was linearity between absorbance and amount of orthophosphate up to 0.16 /Amole at both 713 Abs
Bkc. 4. Absorbance spectra for samples containing O.Og pmole orthophosphate (O), 0.04 @mole orthophosphate plus 2.00 ,umoles pyrophosphate (a) and 2.00 pmoles pyrophosphate (X). All readings were made in 10 mm cuvets against distilled water on a Zeiss PM& II spectrophotometer. Abscissa: wavelength values in nm. Ordinate: absorbance (sample - reagent blank).
42
MOGEKS
H@RDER
Abs.
0.04
0.06
0.12
016 rmol
FIG. 5. Orthophosphate standard curves at wavelengths 713 nm (0) and 850 nm orthophosphate in sample. Ordinate : absorbance (0). Abscissa : inorganic (sample - reagent blank).
nm and 850 nm (Fig. 5). Pyrophosphate in samples gave rise to identical absolute increase in absorbance for all amounts of orthophosphate, 0.080 and 0.120 absolute absorbance units at 713 nm and 850 nm, respectively, for 2.0 pmoles of pyrophosphate. Interference
In a solution containing 0.08 /*mole orthophosphate, absorbance increased in direct proportion to the increasing pyrophosphate concentration until approximately 2.0 mmoles/liter, afterwards falling so that when pyrophosphate concentration was 15 mmolesJliter orthophosphate Abs.
FIG. 6. Interference of inorganic pyrophosphate on orthophosphate assay on samples containing 0.08 ,umole orthophosphate and O-20 amoles inorganic pyrophosphate (0). Pyrophosphate blank samples as references (X). Abscissa: inorganic pyrophosphate in sample.
INORGANIC
PYROPHOSPHATASE
ACTIVITY
43
FIG. 7. Recovery of 0.08 pmole orthophosphnte in samples containing inorganic pyrophosphate (X ), adenosindiphosphate ( q ) , P-glycelophosphate (O), and pof phosphate compounds nitrophenyl phosphate (8). Abscissa: concentrations other than orthophosphate.
could not be recovered after compensating for pyrophosphate blank value (Fig. 6). Other phosphate compounds which are also used as phosphohydrolass substrates interfere likewise with orthophosphate determination. Solutions with adenosine diphosphate, /?-glycerophosphate, and p-nitrophenyl phosphate in concentrations between 0.5 and 20 mmoles/liter caused, au did gyrophosphate, a decrease in the recovery of 0.08 blmole orthophosphate, but such that interference declines in the order mentioned (Fig. 7). In order to clarify further t,he process of pyrophosphatc interference, 10 ,umolespyrophosphate was added to a solution containing 0.06 qole orthophosphate at various times during the orthophosphate analysis procedure. According to the results in Fig. 7, a 10% recovery of orthophosphate could be expected. This was also found when pyrophosphate was added at any time before ammonium molybdate or together with it (Fig. 8). The longer the time lapse from the addition of ammonium molybdate, the less pyrophosphate interfered. Pyrophosphnte did not. interfere at all when added together with arsenite-citrate or later. As acetate buffer is used to lower the hydrogen ion concentration in the supernatant after protein precipitation, the recovery of 0.04 and 0.08 pmole orthophosphate in sodium acetate was investigated (Table 2). Recovery was 100% in the case of t,he acetate buffer concentration of 0.7 mole/liter used, but higher acetate concentrations caused interference. Some inorganic and organic compounds which are employed in the systematic investigation of enzyme activity interfered to varying degrees (Fig. 9). Magnesium sulfate, glucose, and urea did not interfere at all in the concentration range investigated up to 1.0 mole/liter. Borate inter-
44
MOGENS
I
. 0
5
H$RDER
IO
I5
20 min.
FIG. 8. Effect of adding, at different times during orthophosphate assay procedure, 10 amoles inorganic pyrophosphate (X) to samples containing 0.06 pmole orthophosphate. 109% reference value is a sample where no pyrophosphate is added at all. Abscissa: time in orthophosphate procedure. Ordinate: per cent recovery of orthophosphate.
TABLE 2 of Acetate on Orthophosphate
Interference
Assay
Orthophosphate in sample, mmole/liter
Sodium acetate in sample, mole/liter 0.05
0.25
0.50
1.00
1.50
2.00
2.50
0.04 0.08
95 98
99 102
100 100
97 98
91 92
79 77
73 72
Figures indicate percentage recovery of orthophosphate
0
0.1
0.2
0.3
01
in samples.
0.5 molll
FIG. 9. Recovery in per cent of 0.04 pmole orthophosphate in samples containing magnesium sulfate or urea or glucose (e), borate (X ), Tris (A), potassium iodide (‘O), sodium citrate (+ ), and lithium chloride or sodium chloride or potassium chloride (0). Abscissa: concentrations of interfering substances in sample.
INORGANIC
PYROPHOSPHATASE
45
ACTIVITY
fered slightly. Lithium, sodium, and potassium chlorides caused uniform interference with an orthophosphate recovery of approximately 15% at a chloride concentration of 0.4 mole/liter and over. Tris, potassium iodide, and sodium citrate inhibited almost totally with concentrations of 0.5, 0.1, and 0.1 mole/liter, respectively. Final Procedure When hydrolysis has ended, 1 ml of 1.2 N (20%) trichloroacetic acid at 0°C was added to 2 ml of enzyme reaction mixture. After standing for 10 min at O-4”, the mixture was centrifuged for 10 min at o-4”. Then 1 ml of acetic acid-sodium acetate buffer, 2.0 moles/liter, pH 5.0, was added to 2 ml of supernatant; 1 ml of this mixture was taken for orthophosphate analysis, double analysis. At a fixed time, 0.7 ml of 10% ascorbic acid and 3% ammonium molybdate was added, freshly mixed in the proportion 2 + 5; 10 min later 1 ml of 2% sodium arsenite-sodium citrate in 2% (v/v) acetic acid was added. The absorbance was read after 10 min at 700 or 850 nm. Orthophosphate amounts of 0.04 and 0.08 pmole dissolved in a reaction mixture without enzyme were taken as standards. At the same time possible interference will be disclosed. Reliability
of Determination
of Pyrophosphatase
Activity
The sensitivity of the pyrophosphatase assay is maximally that of the orthophosphate analysis. This is approximately 0.0025 pmole orthophosphate, as this amount corresponds to an apparatus reading of 0.010 absolute absorbance unit at 10 mm light path at, 713 nm. Given 2 ml of enzyme activity reaction mixture and a reaction time of 5 min, an enzyme-catalyzed hydrolysis of 0.0005 pmole pyrophosphate per minute can be shown. The accuracy of the enzymic pyrophosphate phosphohydrolysis itself has not been investigated, but is ensured partly by the elimination of the nonenzyme-catalyzed hydrolysis of pyrophosphate caused by trichloroacetic acid and molybdate, partly by the detection of, and compensation for interfcrcnce with the orthophosphate analysis which ensures 100% recovery of orthophosphate. The precision of the analysis is also partly determined by the precision of the orthophosphate analysis. The coefficient of variation in 43 analyses of aqueous orthophosphate solutions carried out over a period of 3 months was 2.5%. The minimum coefficient of variation was 0.7% in 10 repeated analyses all on the same day. The precision of pyrophosphatase determination was investigated by performing 10 consecutive activit,y determinations on the same enzyme solution. Average activity was 155 IUJliter (,umole pyrophosphate X min-l X 1 enzyme-l), standard deviation was 3 III/liter, and the coefficient of variation thus 2.07~. 33 sets
46
MOGENS
H@RDER
of double determinations of enzyme activity on different enzyme material with widely differing activity gave a dispersion of double determinations of the order of 0.0003 IU. DISCUSSION
The fact that enzyme activity is quantitatively expressed by the amount of converted substrate raises two analytical problems: (1) partly that substrate consumption during the whole analytical procedure should be a result of the enzyme’s catalytic activity only, and (2) partly that one has to be able to quantitate the amount of converted substrate accurately, either directly or as increase in product growth. The trichloroacetic acid catalysis accompanying protein precipitation which has been shown here is well known from work with acid-labile compounds (13). A high trichloroacetic acid concentration, temperature at approximately room temperature, and longer reaction time increase protein precipitation ability, but the same conditions increase pyrophosphate hydrolysis at the same time. The conditions chosen are therefore a compromise in which a minimal hydrolysis of pyrophosphate can still take place. The same is true of the proved molybdate-catalyzed hydrolysis of pyrophosphate. It is, however, of vital importance in choosing Baginski and Zak’s method (12) that any hydrolysis of pyrophosphate after the addition of arsenite-citrate is of no importance. The development of absorbance found in pyrophosphate solutions after the addition of arsenite-citrate at different times after molybdate could make one suppose that special pyrophosphate molybdate complexes are formed. This will influence the accuracy of the orthophosphatNe analysis. The uniform absorbance spectra for pyrophosphate and orthophosphate solutions are against this view, but the problem is not completely clear. Control of the possibility of interference which is usual for all methods based on molybdate complex formation (14) is of decisive importance for the accuracy of the orthophosphate analysis. Pyrophosphate interferes at such low concentrations that dilution is necessary when the concentration in the enzyme reaction mixture rises above 4.5 mmoles/ liter, equivalent to 2.0 mmoles/liter in the acetate buffer neutralized supernatant used for the orthophosphate analysis. Pyrophosphate seems to interfere with the formation of molybdate-orthophosphate complexes and so the process is similar to the generally known one (14). Interference due to frequently used buffer components and salts illustrates the necessity of controlling orthophosphate standards as “internal.” This means that standards must be included every time the reaction mixture’s composition is changed. In systematic investigations
INORGANIC
PYROPHOSPHATASE
ACTIVITY
47
of enzyme activity it is, however, especially necessary to be able to show whether buffer or salt effect is an actual fact or a simple analytical phenomenon having nothing to do with enzyme activity at all. In a future article, examples of salt and buffer effects of both types will be given, using the pyrophosphatase assay for determining the enzyme activity in human serum. SUMMARY
Using Baginski and Zak’s (12) method for determining inorganic orthophosphate as a starting point, a number of conditions which influence the accuracy and precision of the determination of pyrophosphatase activity have been shown: nonenzyme-catalyzed acid hydrolysis by protein precipitat,ion agents and molybdate-catalyzed hydrolysis of pyrophosphate together with interference in the determination of orthophosphate by the substrate pyrophosphate and other components from the reaction mixture, Tris, chloride, acetate, citrate, boric acid. With regard to these sources of error, a method is described for determining l)yrophosphatase activity, and its reliability is investigated. REFERENCES 1. FLYNN. R. M., JONES, M. E., .~ND LIPM.~IXN, F. A., J. BioE. Chem. 211, 791 (1954). 2. BAILEY, K., in “Methods of Enzymatic Analysis” (Bergmeyer, H.-U., ed.), pp. 644-647. Academic Press, New York/London, 1963. 3. BLOEMERS. H. P. J., STEPHENSON, M. L., AND ZAMECNII~, P. C., Anal. Biochern. 34, 66 (1970). 4. HEINONEN. J., Anal. Biochem. 37, 32 (1970). 5. FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 66, 375 (1925). 6. BAGINSKI, E. S., FOA, P. P., AND ZAK, B., Clin. Chim. Acta 15, 155 (1967). 7. H~us~ta, K. J., Cb. Chim. Acta 22, 454 (1968). S. WEIL-MALHERBE. H., AND GREEN. R. H., Biochem. J. 49, 286 (1951). 9. PARYIN, R., AND SMITH, R. A., Anal. Biochem. 27, 65 (1969). 10. POSEN, S., Ann. Int. Med. 67, 183 (1967). 11. KORNBERC. A.. in “Horizons in Biochemistry” (Kashn, A., and Pullman, B. cds.), pp. 251-264. Academic Press. New York/London, 1962. 12. BAGINSW E. S., AND ZAK, B., Clin. Chim. Actn 5, 834 (1960). 13. LOWRY, 0. H.. AND LOPEZ, J. A,. J. Biol. Chem. 162, 421 (1946). 14. BOLTZ. D. F.. AND MELLON, M. G., And. Chem. 19, 873 (1947).