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Inorganic pyrophosphatase activity of soils
INORGANIC
PYROPHOSPHATASE
ACTIVITY
OF SOILS
W. A. DICK and M. A. TABATABAI Department
of Agronomy,
Iowa State University, (Accrprrd
25 ilpril
Ames, IA 50011, USA
1977)
simple method to assay inorganic pyrophosphatase activity in soils is described. It involves extraction and calorimetric determination of the orthophosphate (Pi) released when 1 g soil is incubated with buffered (pH 8) pyrophosphate (PPi) solution at 37’C for 5 h. The 1N HZSO, used to extract Pi gives quantitative recovery of Pi added to soils, and the calorimetric method used to determine the Pi extracted in the presence of PPi is specific for Pi. The inorganic pyrophosphatase activity of the six soils studied ranged from 50 to 450 ;tg Pi released ‘g ’ soil.5 h- ‘. Steam sterilization (121 C for 1 h), formaldehyde. fluoride, oxalate, and carbonate inhibtted and toluene. Na’. K+. NH:, Cl-. NO,. NO,. SO:-. and EDTA had no effect on the activity of this enzyme in soils. The initial rates of Pi released obeyed zero-order kinetics. The temperature dependence of the rate constant conformed to the Arrhenius equation up to the point of enzyme inactivation (55-C). The activation energy of pyrophosphatase activity of the six soils studred ranged from 32.5 to 43.2 (av 36.1) kJ,mole-‘. Applrcation of the three linear transformatrons of the Mrchaeh-Menten equation indicated that the h’,,, values of PPi for pyrophosphatase in four soils ranged from 20 to 51 (avg. 35) rnM and that the t/,,,.,, ranged from 130 to X30 (av 500) pg Pi released’g -I soil’ 5 h ‘. Studtes of other properties of inorganic pyrophosphatase activity in soils are reported.
Summary-A
tivity. Hydrolysis of PPi in acid medium with time follows first-order kinetics. Our study is based on a calorimetric method to determine Pi in aqueous solutions containing labile organic and inorganic P compounds (Dick and Tabatabai, 1977). This method involves a rapid formation of heteropoly blue in the presence of ascorbic acidtrichloroacetic acid reagent and complexation of the excess molybdate ions by a citrate-arsenite reagent to prevent further formation of blue color from the Pi derived from hydrolysis of PPi. Application of this method to soils showed that soil inorganic pyrophosphatase activity has reaction properties similar to those reported for catalysis by this enzyme isolated from other sources.
INTRODLICTION
Inorganic pyrophosphatase (pyrophosphate phosphohydrolase. EC 3.6.1.1) is the enzyme that catalyzes the hydrolysis of pyrophosphate (PPi) to orthophosphate (Pi). Pyrophosphatase is widely distributed in nature. Its presence has been reported in bacteria. insects, mammalian tissues, and plants (Feder, 1973). Pyrophosphatase activity in soils has also been reported (Blanchar and Hossner. 1969; Gilliam and Sample. 1968; Hashimoto et cl/., 1969; Hossner and Phillips, 1971: Hughes and Hashimoto, 1971). The activity of this enzyme in soils deserves special attention because its substrate, pyrophosphate, is used as a fertilizer. No accurate assay for the activity of this enzyme in soils is available. The current information of the catalysis brought about by this enzyme in soils is based on studies of extraction of Pi from soils after incubation with PPi (Gilliam and Sample. 1968: Sutton elf ul., 1966). There are three problems associated with the measurement of Pi released by enzymatic hydrolysis during the assay: (i) the Pi released may be sorbed by the soil constituents and therefore not be extracted, (ii) Pi may continue to be hydrolyzed from PPi after extraction from the soil for reasons other than the enzymes (e.g. low pH), and (iii) the presence of PPi may interfere with the measurement of Pi. All these problems must be overcome in any method used to assay the pyrophosphatase activity of soils. Douglas et al. (1976) have reported a method to assay the activity of this enzyme in soils. The method includes the recommendation that the PPi solution be made in water (PPi in water behaves as a buffer pH 10.5) and that the Pi released be extracted with 0.5 N HC1:2 N H,S04. Pyrophosphate hydrolyzes in this extractant within a few minutes, and the straight line obtained for pyrophosphatase activity with incubation time is not necessarily an index of enzyme ac-
MATERIALS
The soils used (Table I) were surface (G-15 cm) samples selected to obtain a range in pH, texture, and organic matter. Before use, each sample was airdried and crushed to pass a 2-mm screen. In the analyses reported in Table 1, pH was determined by a glass electrode (soil:water ratio, 1:2.5), organic C by the method of Mebius (1960) and particle-size distribution by the pipette method of Kilmer and Alexander (1949). Total (Na2C03 fusion) and exchangeable Mg were determined by atomic absorption spectrophotometry. METHOD
TO ASSAY
INORGANIC
PYROPHOSPHATASE
RWpltS
Unless otherwise specified, all compounds used were analytical-grade chemicals. Prepare the reagents as follows: modified universal buffer (MUB) as described by Skujins et a/. (1962); 0.1 N HCI by adding 9 ml cone HCI to 700ml water and diluting to 1 1. with water; PPi solution (50m~) by dissolving 59
W.
60
A.
DICK and M. A.
TABATABAI
Table 1. Analyses of soils
Soil
PH
Clarion Nicollet Webster Okoboji Harps Canisteo
4.6 6.1 6.5 7.0 7.6 7.x
Organic carbon i”,) 2.00 2.73 2.91 5.33 3.14 3.11
Magnesium Exchangeable (me-lOOg_’ soil)
1.65
Sarid
f”J
i”i0
0.329 0.436 0.339
23 29 36
37 34 37
0.500
36
21
0.632
30
31
4.97
0.572
38
35
Place 1 g soil (<2 mm) in a SO-ml plastic centrifuge tube, add 3 ml SOmM PPi solution and swirl the tube for a few seconds to mix the contents. Stopper the tube and incubate it at 37°C. After 5 h, remove the stopper and immediately add 3 ml MUB pH 8.0 and 25 ml 1K H,SO,. Stopper the tube and shake it horizontally in a reciprocal shaker for 3 min. Centrifuge the soil suspension for ?Os at 12,00Orcv.;min, and take 1 ml of the supernatant for Pi analysis (Dick and Tabatabai, 1977). Controls should be performed in each series of analyses to allow for Pi not derived from PPi through pyrophosphatase activity. To perform controls, add 3mI MUB pH 8.0 to 1 g soil and incubate for 5 h. After incubation. add 3 ml 5OmM PPi solution, and immediately add 25 ml I N H,S04 and then extract and analyze for Pi as described above. To determine the f(,,, and 1/;;:,...values of pyrophosphatase, five solutions of PPi were used (10, 15. 20. 40, and 60m.~ soil solution basis). The enzyme activity (t’) values thus obtained (pg. Pi released~g~~ ’ soil.5 hY’) were used to calculate the K,,, and v,,,,, values by using the three linear transformations of the Michaelis-Menten equation. The linear transformations. Lineweaver-Burk plot (1;~ vs I/S), Eadie-Hofstee plot (r vs i,jS), and Hanes--Woolf plot (S!‘r vs S) were plotted (Hofstee, 1952).
AUD
Clay
3.56 3.03 5.1 1 3.04
2.23 g Na4P207. 10HzO (Matheson Coleman & Bell Manufacturing Chemists, Norwood, Ohio) in 20ml of MUB. titrating to pH 8.0 with 0.1 N HCl, and diluting to 100 ml with water (prepare this solution daily): MUB pH 8.0 by titrating 20ml of stock MUB to pH 8.0 with 0.1 N HCI and diluting to 100ml with water; 1 N H,SO, by adding 28 ml cone H,SO, to 700ml water and diluting to I 1. with water; and ascorbic acid--trichloroacetic acid (reagent A), ammonium molybdate (reagent B). and sodium citrate-sodium arse?l;te-glacial acetic acid (reagent C) as described by Dick and Tabatabai (1977). Because of the high concentration of PPi used in this work, the concentrations of reagents B and C were made 1.5 times of those recommended for determination of Pi in other types of aqueous solutions containing labile organic and inorganic P compounds.
RESULTS
Total (“CI
DISCXJSSION
This assay of inorganic pyrophosphatase activity in soils is based on systematic studies of factors affect-
ing extraction and calorimetric determination of the Pi released when a buffered PPi solution is incubated with a soil sample at 37’C. The factors studied included extraction with various reagents of Pi added to soils. type of buffer. buffer pH. concentration of PPi, temperature of incubation. amount of soil, and time of incubation.
The procedure to determine Pi released is based on a rapid formation of heteropoly blue and complexation of the excess molybdate ions to prevent further formation of color derived from hydrolysis of PPi in the acidic medium. The intensity of the heteropoly blue produced was measured spectrophotometrically. Tests indicated that the color is stable in the laboratory light for at least 4 h. The I N H,SOS used for extraction of the Pi released gives quantitative (99.5.mlOO.S~,,) recovery of Pi added to soils. The recovery tests were carried out by adding 50. 750. or 1,500 pg Pi as K2HP04 in 1 ml of solution containing various concentrations of PPi to I g soil and treating the mixture with 2 ml MUB of various pH values and determining the Pi extracted. Controls were included by adding I ml of water instead of the 1 ml of Pi and PPi solution. Results indicated that. by using 1 N H,S04 as an extractant in conjunction with the calorimetric method described. the Pi released by pyrophosphatase activity can be determined quantitatively. Tests with other reagents (e.g. 0.03 s NHIF + 0.025 N HCI. 2 N H,SO,). however. showed that these reagents either led to incomplete extraction of the Pi or caused hydrolysis of the PPi added. We found that the rate of hydrolysis of PPi in acid medium increases with increase in concentration of the acid extractant and with increase in shaking time. We were unable to obtain reproducible results with H$O, or WC1 at concentrations above 1 K and shaking time greater than 5 min. Douglas rt trl. (1976) used a mixture of I-ICI and HzS04 (2.5ti) and a shaking time of 15min. To minimize hydrolysis of PPi during extraction, we centrifuged the incubation tube for 30 s and determined the Pi released immediately by analyzing I ml of the supernatant. Filtration of the soil suspension as recommended by Douglas rr trl. (iY76) takes time and may lead to hydrolysis of PPi present in the soil extract. The control is designed so that it allows for subtraction of native soil Pi and any trace amount of Pi produced by chemical hydrolysis of PPi in the acid medium durin,g the 3 min of the extraction step. No immobilization of Pi was detected when the soils were
Pyrophosphatase
500
activity
of soils
600-
I, Y) 400 = :: ‘; 300 = b. 2. z 2
200
: cz .IP
loo 0
61
. WEBSTER SOIL 0 CLARION SOIL A OKOBOJI SOIL A HARPS SOIL
. WEBSTER SOIL 0 NICOLLET SOIL A OKOBOJI SOIL I
I
I
I
I
I
5
6
7
a
9
10
0
12
pH OF BUFFER
Fig.
1. Effect of pH of buffer on release of Pi in assay
of soil pyrophosphatase
activity.
incubated with Pi under the assay procedure described. Experiments showed that PPi is stable in buffer adjusted to pH 3-10 and incubated at 37’C for 24 h. Choice of’ b@er
and hufer
pH
Among the buffers tested were citrate, acetate, THAM, and modified universal buffers. Citrate and acetate are weak buffers, and it was difficult to maintain the soil-buffer mixture close to the pH desired. The results with THAM and universal buffers were similar, but the universal buffer used has a strong buffering poise and can be used over a wide range of pH (3-12) and was selected for this work. Choice of buffer pH was based on studies showing that maximum activity occurs with MUB pH 8.0. Figure 1 shows results obtained with three soils differing in pH from 6.2 to 7.0 in studies of effect of varying pH of MUB. The other three soils used showed optimum activity at a similar buffer pH. The observed optimum pH for pyrophosphatase activity in soils is similar to that (7.5585) of pyrophosphatase purified from
700-
3
4
5
6
1
INCUBATION TIME(h)
Fig. 3 Effect of time of incubation assay of so11 pyrophosphatase
Ferrobacillus
qf‘ soil
and
of Pi in
(Howard and Lundgren, of yeast (Butler, 1971).
jkrroo.xidans
1970) and that (7.48.1) Amount
on release activity.
toluene
The observed linear releationship between amount of soil and amount of Pi released (Fig. 2) is further evidence that the method described measures pyrophosphatase activity and that neither the PPi concentration nor the amount of Pi formed influence the reaction velocity of pyrophosphatase. Results were not significantly affected when toluene was added nor when the amount of toluene was increased from 0.25 to 2ml. It is often necessary to inhibit microbial growth and assimilation of enzymatic reaction products in assay of soil enzyme activity. Toluene has been used for this purpose. Several workers have found that toluene affects the activity of some enzymes in soils (Skujins, 1967), but we found that it did not have any effect on pyrophosphatase activity of soils. Skujins (1967) has reviewed the literature on the effects of toluene on enzyme activity and discussed the use of toluene to unmask enzymes in
1
. WEBSTER SOIL CLARION SOIL r HARPS SOIL A
600-
0
0.5 AMOUNT
1.0
1.5
OF SOIL (gl
Ftg. 2. Effect of amount of soil on release of Pi in assay of soil pyrophosphatase activity.
I
I
I
I
I
40
50
60
70
INCUBATION TEMPERATURE
Fig. 4. Effect of temperature of incubation Pi in assay of soil pyrophosphatase
80
(Cl
on release activity.
of
62
W. A. D~cx and M. .4. TARATABAI
. 0 A *
used for correction of activity of unsterilized soils. The rate of chemical hydrolysis of PPi at temperatures below 40 C is not significant. but it increases as the incubation temperature increases. This chemical hydrolysis of PPi with temperature. hoaecer. is reproducible. The rates of chemical hydrolysis of PPi were almost identical between 40 and 80 C in the six soils studied. Pyrophosphatase acti\ ity was assayed at an incubation temperature of 37 C because this temperature has been used extensively for assay of pyrophosphatase and other enzyme activities and also because preliminary work showed that it was not necessary to use ;I higher temperature to obtain precise results of pyrophosphatase activity by this method. Also. this temperature was selected because PPi is chemically hydrolyzed at temperatures above 40 C. and special controls should be included. which makes the assay procedure dilticult.
CLARION SOIL OKOBOJI SOIL HARPS SOIL CANISTEO SOIL
-1 3.1
3.2
3.3
3.4
3.5
l/T("K-'X103)
Fig. 5. Arrhenius equation plot of pyrophosphatase tivity values of Clarion. Okoboji, Harps and Canisteo
acsoils.
assay of enzymatic activities of microorganisms. This unmasking effect seems to be due to the ability of toluene to render microbial cell membranes permeable to substrates and enzyme reaction products. From our results, it seems either the microbial cell membranes are permeable to PPi and Pi and that toluene therefore has no significant effect on the activity of this enzyme in soils. or the pyrophosphatase activity associated with the microorganisms in soils is an insignificant part of the total activity.
The rate of enzyme-catalyzed reactions increases as the temperature increases until some temperature at which the rate begins to decrease because of inactivation of the enzyme (Fig. 4). The temperature dependence of the rate constant at lower temperatures (below temperature of inactivation) can be described by the Arrhenius equation: I< = .4 exp ( - Ec(,‘R T) where I< is the rate constant, A is the pre-exponential factor, Err is the activation ener_gy. R is the gas constant, and Tis the temperature m K. The Arrhenius equation also can be expressed in the log form: log !i = (- Ed?.303
Enzyme reactions normally give a straight-line relationship between amount of reaction product and time of incubation. Figure 3 shows results obtained in studies of the effect of varying the time of incubation on the Pi released. Because the substrate (PPi) is hydrolyzed in acid solutions. the controls were designed to detect any such hydrolysis. Therefore. the observed linear relationship between reaction time and amount of Pi released is the true property of pyrophosphatase activity in soils. Formation of Pi in the soils studied was a zero-order reaction for at least 7 h. With longer incubation, however. the reaction velocity deviated from linearity. indicating either that the PPI was limiting or that the Pi released was inhibiting the reaction rate catalyzed by pyrophosphatase. Studies of the effect of temperature on pyrophosphatase activity of soils showed that inactivation of pyrophosphatase in soils occurs at about 55’C (Fig. 4). The inactivation temperature of pyrophosphatase is about IO C lower than that of sulfatase (Tabatabai and Bremner, 1’370) and rhodanese (Tabatabai and Singh, 1976) activities in soils. Temperature has a significant effect on chemical hydrolysis of PPi. The experiments for effect of temperature on pyrophosphatase activity in soils were carried out with appropriate controls. For controls, pyrophosphatase activity was assayed in steam-sterilized soils as described in the assay procedure, and the amount of Pi released by pyrophosphatase activity at each temperature was
RT) + log A.
The activation energy can be calculated from a plot of log I\ or log of initial rate vs l;7: The Arrhenius plot for pyrophosphatase activity ~alucs (Llg Pi released,g- ’ soil.5 h-’ ) in the soil studied were linear between 10 and 50 C (Fig. 5). The slopes of the four lines in Fig. 5 and, therefore, the energies of activation of the reaction catalyzed by the pyrophosphatase in the four soils (Clarion, Okoboji, Harps and Canisteo) were almost identical. The Arrhenius plots of the results obtained with the other two soils studied showed similar slopes. indicating similar values of activation energy for pyrophosphatasc in all the soils used. The energy of activation of the reaction catalyzed by pyrophosphatase in the siu soils studied. expressed in kJ mol I. ranged from 32.5 to 33.2 (a\ 36.1). calculated from Arrhcnius plots similar to those shown in Fig. 5. The range of activation energies of soil pyrophosphatase agrees well with the range (34.X 31 .OkJ mole- I I for potato pyrophosphatase (Naganna ct rd., 1YSS). Energy of activation \,alue of about 3Y.XkJ.mole- ’ was found by Kunitr (lY52) for yeast pyrophosphatase.
For valid assay of enzymatic activity, it is necessary to ensure that the substrate concentration is not limiting the reaction velocity in the assay procedure. A study of the effect of varying the substrate (PPi) concentration (Fig. 6) showed that the concentration
Pyrophosphatase
of soils
63
(7 CLARION SO
100
0
activity
I
20
,
I
I
I
,
40 60 80 100 120 PPi CONCENTRATION fmMl
6. Effect of substrate
(PPi)
concentration
of Pi in assay of soil pyrophosphatase
140
on release
activity.
adopted (50m~) was satisfactory. At a PPi concentration above 60m~, however, the enzyme reaction velocity decreased. The magnitude of this decrease was greater as the PPi concentration increased. This lower rate of PPr hydrolysis at high concentration seems due to two factors. One is that, at high concentrations, the substrate (free PPi) is known to inhibit pyrophosphatase, and the other is that Mg” is required for activation of PPi for binding with the enzyme (Butler, 1971). The results (Fig. 6) indicate either that, at high PPi concentrations, not enough Mg2 + was present in the soils to produce enough mono-magnesium PPi complex, the substrate for this enzyme, or that PPi inhibited pyrophosphatase. With the exception of one soil (Clarion), the soils used contained similar amounts of exchangeable Mg*+ (Table 1); therefore, the rate-limiting step with increasing the PPi concentration was observed at similar PPi concentration (Fig. 6). Similar curves have been reported by Howard and Lundgren (1970) for pyrophosphatase of F. ,~e~~~o~i~aFls. Michaelis-Menten
constant
Figure 7 shows plots of the three possible linear transformations of the Michaelis-Menten equation
Table 2. K,
Fig. 7. The three possible linear plots of the MichaelisMenten equation. Velocity (u) is expressed as pg Pi released.g-’ soil’ 5 h-r and S is in M. A, Lineweaver-Burk plot (l/v vs l/S); B, Eadie-Hofstee plot (G vs u/S); C, HanesWoolf plot (S/v vs S). n, Okoboji soil; c7, Clarion soil; l, Webster soil.
applied to the pyrophosphatase activity values obtained as a function of PPi concentration. To eliminate the rate-limiting effect of Mg2’ and the inhibition by PPi, *we plotted the activity values obtained with up to 60m~ PPi. The straight lines shown in Fig. 7 are those calculated by regression analysis. As it can be seen from Fig. 7, the results obtained obeyed the three linear transformations of the MichaelisMenten equation.
and F$,.., values of inorganic pyrophosphatase the three transformations of MichaehsMenten
Michaelis-Menten transformation Lineweaver-Burk (l/v vs l/S?
Plot
Soil
K (mM)
Webster Clarion Okoboji Harps
43 51 36 21 38 36 4.5 35 20 35 35 45 34 20 34
Average Eadie-Hofstee (1!vs u/S)
Webster Clarion Okoboji Harps
Plot
Average Hanes-Woolf (Sir vs S)
Webster Clarion Okoboji Harps
Plot
Average *pg Pi released.g-’
soil.5 h-l.
in soils calculated equation
from
I/lndX* 826 730 429 131 529 743 672 422 128 491 730 649 411 130 482
64
w.
A. DICK and ,u, A.
Table 3. Precision
Soil
No. of analyses
Clarion 6 Nicoilet 7 Webster 6 Okoboji 8 Harps 6 Cnnisteo 7 * /tg Pi released.gvariation.
+
Nom Formaldehyde (0.25 ml) Steam sterilization (121 c, 1 h) NaF NazCzO, Na&O,
of method
Pyrophosphatase Mean
Range 33s-337 422432 388-402 220 233 76-X 5 2-60
treatments
activity* SD
CV
1.5 4.5 5.3 6.3 1.6 3.0
0.8 1.1 1.5 1.8 2.1 5.5
335 428 393 227 78 55
’ :ml-S h- ‘. SD, standard
Table 4. Effects of various
Soil treatment
TABATAtlAl
deviation:
CV. coefficient
on pyrophosphatase
Pyrophosphatase activity Nicollet Clarion
activity
of
of soils
of soil specified* Okoboji Harps
335 1’4 17
428 I63 11
227 57 I
78 45 8
24x 701 124
398 321 227
200 129 120
46 4s 4x
* /~g Pi re1eased.g ’ soil.5 h-‘. + A sample of soil (I g) was treated with 0.25 ml of solution made 0.2 M with respect to the inorganic compound specified. After 30 min of equilibration at room temperature (22’C). pyrophosphatase activity was assayed. Na,EDTA. NaCI, N&Cl, KCl, NaNO,, NaNO,: Na*SO,. and toluene (0.25 ml) had no effect,
The K,,, and V,,,:,, values calculated from the plots in Fig. 7 are shown in Table 2. The K,,, values of PPi for pyrophosphatase in four soils ranged from 20 to 50 mM (avg. 35 mM) and the r/;,,‘,, values ranged from 130 to 830 (av SOO) 118 Pi released.g-’ soil 5 h- ‘. The K,,, values of PPi for pyropllosphatase in soils is about one order of magnitude greater than that reported for pyrophosphatase purified from F. ftrrosidn~s (Howard and Lundgren, 1970). This high in apparent K,, value of PPi for pyrophosphatase soils compared with that for the same enzyme of other sources is partly due to sorption of PPi by soil ConstitLIents. The conformity of the reaction rates to the three linear transformations of the Michaelis-Menten equation (Fig. 7). however. suggests that sorption of PPi by soils is proportional to the PPi concentration used.
The pyrophosph~ltase activity values of six soils (Tables 1 and 3) ranged from 55 to 42X (av 253) pg Pi re1eased.g’ soil 5 h ‘. and the standard deviation of the activity determinations ranged from 1.6 to 5.3 (av 3.9). The high precision of the method results largely because extraction and determination of the Pi released is quantitative. and the procedures are simple and readily standardized.
Steam sterilization is known to inactivate soil enzymes, but the results obtained after this treatment
showed some apparent pyrophosphatase activity (Table 4). These apparent activities after steam sterilization, however, are much less than those of the untreated soils. The pyrophosphatase activity after heat treatment could be due to nonspecific chemical hydrolysis of PPi by metal ions. Expressed as a percentage of total pyrophosphatase activities in unsterilized soils, the apparent activities after steam sterilization ranged from 2.6”; for Nicollet soil to 10.3”, for Harps soil. Treatment of soils with formaldehyde, Iluoride. oxalate. and carbonate inhibited pyrophosphatase activity. This inhibition seems to be related to binding of PPi (3utler. of the Mg2+ required for activation 1971). Among the chemical treatments studied, toluene, Na +, K+, NH,‘. Cl-. NO,. NOz. SO:-, and EDTA showed no effect on the activity of this enzyme in soils. ilcXiin~~t~~lget?lcntsJournal Paper J-8690 of the lo%:t Agriculture anti Home Economics Experiment Station, Ames, Iowa. Projects
1868. 2082 and 2112.
REFERENCXS
R. W. and H~SSNER L. R. (1969) Hydrolysis and sorption of ortho. pyro, tripoly and trimetaphosphate in 32 midwestern soils. Proc. Soil Sci. SIC. .AM. 33, 622-625. BUTL~K L. G. (1971) Yeast and other inorganic pyrophosphatases. In 7hr En~~?nes (P. D. Boyer. ed.) Vol. 4. pp. 529-541. Academic Press. New York. BLANCHAK
Pyrophosphatase
DICK W. A. and TABATABAI M. A. (1977) Determination of orthophosphate in aqueous solutions containing labile organic and inorganic phosphorus compounds. J. En1+rotz. ouui. 6, 82-85. Dowx.~
and D. T. Mitchell, Eds.) pp. 475-508. John Wiley, New York. GILLIAMJ. W. and SAMPLEE. C. (1968) Hydrolysis of pyrophosphate in soils: pH and biological effects. Soii Sci. 106, 352- 357. HASHI~~~TO I., HUGHESJ. D., and PIUI.EN0. D. JR. (1969) Reaction of triammonium pyrophosphate with soils and soil minerals. hoc. Soil Sci. Sot. Am. 33, 401405. HOFSTEEH. J. (1952) On the evaluation of the constants G, and K,,, in enzyme reactions. Science 116. 329331. H~~SSNER L. R. and PHILLIPSD. P. (1971) ~rophosphat~ hydrolysis in Aooded soil. Proc. Soil Sri. Sot. Am. 35, 379 ~383. HOWARDA. and LUNUGREXD. G. (1970) Inorganic pyrophosphatase from Ferrobacillus ,ferrooxidans (Thiohcillus ferrooxidms).
Can. J. Biochem.
48, 1302-1307.
activity
of soils
65
HUGHESJ. D. and HASHIMOTOI. (1971) Triammonium pyrophosphate as a source of phosphorus for plants. Proc. Soil. Sci. Sot. Am. 35, 643-647. KILMERV. J. and ALEXANDER L. 7. (1949) Methods of making mechanical analyses of soils. Soii Sci. 48, 15-24. K~NITZ M. (1952) Crystalline inorganic pyrophosphatase isolated from baker’s yeast. J. germ.PhJsiof. 35, 423-450. MERIUSL. J. (1960) A rapid method for the determination of organic carbon in soil. Alzrxlytica ckirn. Acta 22, 120-124. NAGANNAB., RAMANA., VEE~UGOPAL B., and SRIPATHIC. E. (1955) Potato pyrophosphatases, ~i~~c~e~. J. 60, 215-223. SKUJINSJ. J. (1967) Enzymes in soil. In Soil ~joc~e~?~isfr~ (A. D. McLaren and G. H. Peterson. Eds.) Vol. 1, pp. 371-414. Marcel Dekker. New York. SKUJINSJ. J., BRAALL., and MCLAKENA. D. (1962) Characterization of phosphatase in a terrestrial soil sterilized with an electron beam. Enzrmolouia 25, 125-133. SUTTONC. D.. GLJNARYD., and LARSENS. (1966) Pyrophosphate as a source of phosphorus for plants-II. &ydrolysis and initial uptake by a barley crop. Soil Sci, 101; 199-204.
_
-
.
.
TABA~ABAI M. A. and BREMNER J. M. (1970) Arylsulfatase activity of soils. Proc. Soil Sci. Sm. Am. 34, 225-229. TABATABA~M. A. and SINGH B. 8. (1976) Rhodanese activity of soils. Soil Sci. Sot. AWL 1. 40, 381-385.
PYROPHOSPHATASE
ACTIVITY
OF SOILS
W. A. DICK and M. A. TABATABAI Department
of Agronomy,
Iowa State University, (Accrprrd
25 ilpril
Ames, IA 50011, USA
1977)
simple method to assay inorganic pyrophosphatase activity in soils is described. It involves extraction and calorimetric determination of the orthophosphate (Pi) released when 1 g soil is incubated with buffered (pH 8) pyrophosphate (PPi) solution at 37’C for 5 h. The 1N HZSO, used to extract Pi gives quantitative recovery of Pi added to soils, and the calorimetric method used to determine the Pi extracted in the presence of PPi is specific for Pi. The inorganic pyrophosphatase activity of the six soils studied ranged from 50 to 450 ;tg Pi released ‘g ’ soil.5 h- ‘. Steam sterilization (121 C for 1 h), formaldehyde. fluoride, oxalate, and carbonate inhibtted and toluene. Na’. K+. NH:, Cl-. NO,. NO,. SO:-. and EDTA had no effect on the activity of this enzyme in soils. The initial rates of Pi released obeyed zero-order kinetics. The temperature dependence of the rate constant conformed to the Arrhenius equation up to the point of enzyme inactivation (55-C). The activation energy of pyrophosphatase activity of the six soils studred ranged from 32.5 to 43.2 (av 36.1) kJ,mole-‘. Applrcation of the three linear transformatrons of the Mrchaeh-Menten equation indicated that the h’,,, values of PPi for pyrophosphatase in four soils ranged from 20 to 51 (avg. 35) rnM and that the t/,,,.,, ranged from 130 to X30 (av 500) pg Pi released’g -I soil’ 5 h ‘. Studtes of other properties of inorganic pyrophosphatase activity in soils are reported.
Summary-A
tivity. Hydrolysis of PPi in acid medium with time follows first-order kinetics. Our study is based on a calorimetric method to determine Pi in aqueous solutions containing labile organic and inorganic P compounds (Dick and Tabatabai, 1977). This method involves a rapid formation of heteropoly blue in the presence of ascorbic acidtrichloroacetic acid reagent and complexation of the excess molybdate ions by a citrate-arsenite reagent to prevent further formation of blue color from the Pi derived from hydrolysis of PPi. Application of this method to soils showed that soil inorganic pyrophosphatase activity has reaction properties similar to those reported for catalysis by this enzyme isolated from other sources.
INTRODLICTION
Inorganic pyrophosphatase (pyrophosphate phosphohydrolase. EC 3.6.1.1) is the enzyme that catalyzes the hydrolysis of pyrophosphate (PPi) to orthophosphate (Pi). Pyrophosphatase is widely distributed in nature. Its presence has been reported in bacteria. insects, mammalian tissues, and plants (Feder, 1973). Pyrophosphatase activity in soils has also been reported (Blanchar and Hossner. 1969; Gilliam and Sample. 1968; Hashimoto et cl/., 1969; Hossner and Phillips, 1971: Hughes and Hashimoto, 1971). The activity of this enzyme in soils deserves special attention because its substrate, pyrophosphate, is used as a fertilizer. No accurate assay for the activity of this enzyme in soils is available. The current information of the catalysis brought about by this enzyme in soils is based on studies of extraction of Pi from soils after incubation with PPi (Gilliam and Sample. 1968: Sutton elf ul., 1966). There are three problems associated with the measurement of Pi released by enzymatic hydrolysis during the assay: (i) the Pi released may be sorbed by the soil constituents and therefore not be extracted, (ii) Pi may continue to be hydrolyzed from PPi after extraction from the soil for reasons other than the enzymes (e.g. low pH), and (iii) the presence of PPi may interfere with the measurement of Pi. All these problems must be overcome in any method used to assay the pyrophosphatase activity of soils. Douglas et al. (1976) have reported a method to assay the activity of this enzyme in soils. The method includes the recommendation that the PPi solution be made in water (PPi in water behaves as a buffer pH 10.5) and that the Pi released be extracted with 0.5 N HC1:2 N H,S04. Pyrophosphate hydrolyzes in this extractant within a few minutes, and the straight line obtained for pyrophosphatase activity with incubation time is not necessarily an index of enzyme ac-
MATERIALS
The soils used (Table I) were surface (G-15 cm) samples selected to obtain a range in pH, texture, and organic matter. Before use, each sample was airdried and crushed to pass a 2-mm screen. In the analyses reported in Table 1, pH was determined by a glass electrode (soil:water ratio, 1:2.5), organic C by the method of Mebius (1960) and particle-size distribution by the pipette method of Kilmer and Alexander (1949). Total (Na2C03 fusion) and exchangeable Mg were determined by atomic absorption spectrophotometry. METHOD
TO ASSAY
INORGANIC
PYROPHOSPHATASE
RWpltS
Unless otherwise specified, all compounds used were analytical-grade chemicals. Prepare the reagents as follows: modified universal buffer (MUB) as described by Skujins et a/. (1962); 0.1 N HCI by adding 9 ml cone HCI to 700ml water and diluting to 1 1. with water; PPi solution (50m~) by dissolving 59
W.
60
A.
DICK and M. A.
TABATABAI
Table 1. Analyses of soils
Soil
PH
Clarion Nicollet Webster Okoboji Harps Canisteo
4.6 6.1 6.5 7.0 7.6 7.x
Organic carbon i”,) 2.00 2.73 2.91 5.33 3.14 3.11
Magnesium Exchangeable (me-lOOg_’ soil)
1.65
Sarid
f”J
i”i0
0.329 0.436 0.339
23 29 36
37 34 37
0.500
36
21
0.632
30
31
4.97
0.572
38
35
Place 1 g soil (<2 mm) in a SO-ml plastic centrifuge tube, add 3 ml SOmM PPi solution and swirl the tube for a few seconds to mix the contents. Stopper the tube and incubate it at 37°C. After 5 h, remove the stopper and immediately add 3 ml MUB pH 8.0 and 25 ml 1K H,SO,. Stopper the tube and shake it horizontally in a reciprocal shaker for 3 min. Centrifuge the soil suspension for ?Os at 12,00Orcv.;min, and take 1 ml of the supernatant for Pi analysis (Dick and Tabatabai, 1977). Controls should be performed in each series of analyses to allow for Pi not derived from PPi through pyrophosphatase activity. To perform controls, add 3mI MUB pH 8.0 to 1 g soil and incubate for 5 h. After incubation. add 3 ml 5OmM PPi solution, and immediately add 25 ml I N H,S04 and then extract and analyze for Pi as described above. To determine the f(,,, and 1/;;:,...values of pyrophosphatase, five solutions of PPi were used (10, 15. 20. 40, and 60m.~ soil solution basis). The enzyme activity (t’) values thus obtained (pg. Pi released~g~~ ’ soil.5 hY’) were used to calculate the K,,, and v,,,,, values by using the three linear transformations of the Michaelis-Menten equation. The linear transformations. Lineweaver-Burk plot (1;~ vs I/S), Eadie-Hofstee plot (r vs i,jS), and Hanes--Woolf plot (S!‘r vs S) were plotted (Hofstee, 1952).
AUD
Clay
3.56 3.03 5.1 1 3.04
2.23 g Na4P207. 10HzO (Matheson Coleman & Bell Manufacturing Chemists, Norwood, Ohio) in 20ml of MUB. titrating to pH 8.0 with 0.1 N HCl, and diluting to 100 ml with water (prepare this solution daily): MUB pH 8.0 by titrating 20ml of stock MUB to pH 8.0 with 0.1 N HCI and diluting to 100ml with water; 1 N H,SO, by adding 28 ml cone H,SO, to 700ml water and diluting to I 1. with water; and ascorbic acid--trichloroacetic acid (reagent A), ammonium molybdate (reagent B). and sodium citrate-sodium arse?l;te-glacial acetic acid (reagent C) as described by Dick and Tabatabai (1977). Because of the high concentration of PPi used in this work, the concentrations of reagents B and C were made 1.5 times of those recommended for determination of Pi in other types of aqueous solutions containing labile organic and inorganic P compounds.
RESULTS
Total (“CI
DISCXJSSION
This assay of inorganic pyrophosphatase activity in soils is based on systematic studies of factors affect-
ing extraction and calorimetric determination of the Pi released when a buffered PPi solution is incubated with a soil sample at 37’C. The factors studied included extraction with various reagents of Pi added to soils. type of buffer. buffer pH. concentration of PPi, temperature of incubation. amount of soil, and time of incubation.
The procedure to determine Pi released is based on a rapid formation of heteropoly blue and complexation of the excess molybdate ions to prevent further formation of color derived from hydrolysis of PPi in the acidic medium. The intensity of the heteropoly blue produced was measured spectrophotometrically. Tests indicated that the color is stable in the laboratory light for at least 4 h. The I N H,SOS used for extraction of the Pi released gives quantitative (99.5.mlOO.S~,,) recovery of Pi added to soils. The recovery tests were carried out by adding 50. 750. or 1,500 pg Pi as K2HP04 in 1 ml of solution containing various concentrations of PPi to I g soil and treating the mixture with 2 ml MUB of various pH values and determining the Pi extracted. Controls were included by adding I ml of water instead of the 1 ml of Pi and PPi solution. Results indicated that. by using 1 N H,S04 as an extractant in conjunction with the calorimetric method described. the Pi released by pyrophosphatase activity can be determined quantitatively. Tests with other reagents (e.g. 0.03 s NHIF + 0.025 N HCI. 2 N H,SO,). however. showed that these reagents either led to incomplete extraction of the Pi or caused hydrolysis of the PPi added. We found that the rate of hydrolysis of PPi in acid medium increases with increase in concentration of the acid extractant and with increase in shaking time. We were unable to obtain reproducible results with H$O, or WC1 at concentrations above 1 K and shaking time greater than 5 min. Douglas rt trl. (1976) used a mixture of I-ICI and HzS04 (2.5ti) and a shaking time of 15min. To minimize hydrolysis of PPi during extraction, we centrifuged the incubation tube for 30 s and determined the Pi released immediately by analyzing I ml of the supernatant. Filtration of the soil suspension as recommended by Douglas rr trl. (iY76) takes time and may lead to hydrolysis of PPi present in the soil extract. The control is designed so that it allows for subtraction of native soil Pi and any trace amount of Pi produced by chemical hydrolysis of PPi in the acid medium durin,g the 3 min of the extraction step. No immobilization of Pi was detected when the soils were
Pyrophosphatase
500
activity
of soils
600-
I, Y) 400 = :: ‘; 300 = b. 2. z 2
200
: cz .IP
loo 0
61
. WEBSTER SOIL 0 CLARION SOIL A OKOBOJI SOIL A HARPS SOIL
. WEBSTER SOIL 0 NICOLLET SOIL A OKOBOJI SOIL I
I
I
I
I
I
5
6
7
a
9
10
0
12
pH OF BUFFER
Fig.
1. Effect of pH of buffer on release of Pi in assay
of soil pyrophosphatase
activity.
incubated with Pi under the assay procedure described. Experiments showed that PPi is stable in buffer adjusted to pH 3-10 and incubated at 37’C for 24 h. Choice of’ b@er
and hufer
pH
Among the buffers tested were citrate, acetate, THAM, and modified universal buffers. Citrate and acetate are weak buffers, and it was difficult to maintain the soil-buffer mixture close to the pH desired. The results with THAM and universal buffers were similar, but the universal buffer used has a strong buffering poise and can be used over a wide range of pH (3-12) and was selected for this work. Choice of buffer pH was based on studies showing that maximum activity occurs with MUB pH 8.0. Figure 1 shows results obtained with three soils differing in pH from 6.2 to 7.0 in studies of effect of varying pH of MUB. The other three soils used showed optimum activity at a similar buffer pH. The observed optimum pH for pyrophosphatase activity in soils is similar to that (7.5585) of pyrophosphatase purified from
700-
3
4
5
6
1
INCUBATION TIME(h)
Fig. 3 Effect of time of incubation assay of so11 pyrophosphatase
Ferrobacillus
qf‘ soil
and
of Pi in
(Howard and Lundgren, of yeast (Butler, 1971).
jkrroo.xidans
1970) and that (7.48.1) Amount
on release activity.
toluene
The observed linear releationship between amount of soil and amount of Pi released (Fig. 2) is further evidence that the method described measures pyrophosphatase activity and that neither the PPi concentration nor the amount of Pi formed influence the reaction velocity of pyrophosphatase. Results were not significantly affected when toluene was added nor when the amount of toluene was increased from 0.25 to 2ml. It is often necessary to inhibit microbial growth and assimilation of enzymatic reaction products in assay of soil enzyme activity. Toluene has been used for this purpose. Several workers have found that toluene affects the activity of some enzymes in soils (Skujins, 1967), but we found that it did not have any effect on pyrophosphatase activity of soils. Skujins (1967) has reviewed the literature on the effects of toluene on enzyme activity and discussed the use of toluene to unmask enzymes in
1
. WEBSTER SOIL CLARION SOIL r HARPS SOIL A
600-
0
0.5 AMOUNT
1.0
1.5
OF SOIL (gl
Ftg. 2. Effect of amount of soil on release of Pi in assay of soil pyrophosphatase activity.
I
I
I
I
I
40
50
60
70
INCUBATION TEMPERATURE
Fig. 4. Effect of temperature of incubation Pi in assay of soil pyrophosphatase
80
(Cl
on release activity.
of
62
W. A. D~cx and M. .4. TARATABAI
. 0 A *
used for correction of activity of unsterilized soils. The rate of chemical hydrolysis of PPi at temperatures below 40 C is not significant. but it increases as the incubation temperature increases. This chemical hydrolysis of PPi with temperature. hoaecer. is reproducible. The rates of chemical hydrolysis of PPi were almost identical between 40 and 80 C in the six soils studied. Pyrophosphatase acti\ ity was assayed at an incubation temperature of 37 C because this temperature has been used extensively for assay of pyrophosphatase and other enzyme activities and also because preliminary work showed that it was not necessary to use ;I higher temperature to obtain precise results of pyrophosphatase activity by this method. Also. this temperature was selected because PPi is chemically hydrolyzed at temperatures above 40 C. and special controls should be included. which makes the assay procedure dilticult.
CLARION SOIL OKOBOJI SOIL HARPS SOIL CANISTEO SOIL
-1 3.1
3.2
3.3
3.4
3.5
l/T("K-'X103)
Fig. 5. Arrhenius equation plot of pyrophosphatase tivity values of Clarion. Okoboji, Harps and Canisteo
acsoils.
assay of enzymatic activities of microorganisms. This unmasking effect seems to be due to the ability of toluene to render microbial cell membranes permeable to substrates and enzyme reaction products. From our results, it seems either the microbial cell membranes are permeable to PPi and Pi and that toluene therefore has no significant effect on the activity of this enzyme in soils. or the pyrophosphatase activity associated with the microorganisms in soils is an insignificant part of the total activity.
The rate of enzyme-catalyzed reactions increases as the temperature increases until some temperature at which the rate begins to decrease because of inactivation of the enzyme (Fig. 4). The temperature dependence of the rate constant at lower temperatures (below temperature of inactivation) can be described by the Arrhenius equation: I< = .4 exp ( - Ec(,‘R T) where I< is the rate constant, A is the pre-exponential factor, Err is the activation ener_gy. R is the gas constant, and Tis the temperature m K. The Arrhenius equation also can be expressed in the log form: log !i = (- Ed?.303
Enzyme reactions normally give a straight-line relationship between amount of reaction product and time of incubation. Figure 3 shows results obtained in studies of the effect of varying the time of incubation on the Pi released. Because the substrate (PPi) is hydrolyzed in acid solutions. the controls were designed to detect any such hydrolysis. Therefore. the observed linear relationship between reaction time and amount of Pi released is the true property of pyrophosphatase activity in soils. Formation of Pi in the soils studied was a zero-order reaction for at least 7 h. With longer incubation, however. the reaction velocity deviated from linearity. indicating either that the PPI was limiting or that the Pi released was inhibiting the reaction rate catalyzed by pyrophosphatase. Studies of the effect of temperature on pyrophosphatase activity of soils showed that inactivation of pyrophosphatase in soils occurs at about 55’C (Fig. 4). The inactivation temperature of pyrophosphatase is about IO C lower than that of sulfatase (Tabatabai and Bremner, 1’370) and rhodanese (Tabatabai and Singh, 1976) activities in soils. Temperature has a significant effect on chemical hydrolysis of PPi. The experiments for effect of temperature on pyrophosphatase activity in soils were carried out with appropriate controls. For controls, pyrophosphatase activity was assayed in steam-sterilized soils as described in the assay procedure, and the amount of Pi released by pyrophosphatase activity at each temperature was
RT) + log A.
The activation energy can be calculated from a plot of log I\ or log of initial rate vs l;7: The Arrhenius plot for pyrophosphatase activity ~alucs (Llg Pi released,g- ’ soil.5 h-’ ) in the soil studied were linear between 10 and 50 C (Fig. 5). The slopes of the four lines in Fig. 5 and, therefore, the energies of activation of the reaction catalyzed by the pyrophosphatase in the four soils (Clarion, Okoboji, Harps and Canisteo) were almost identical. The Arrhenius plots of the results obtained with the other two soils studied showed similar slopes. indicating similar values of activation energy for pyrophosphatasc in all the soils used. The energy of activation of the reaction catalyzed by pyrophosphatase in the siu soils studied. expressed in kJ mol I. ranged from 32.5 to 33.2 (a\ 36.1). calculated from Arrhcnius plots similar to those shown in Fig. 5. The range of activation energies of soil pyrophosphatase agrees well with the range (34.X 31 .OkJ mole- I I for potato pyrophosphatase (Naganna ct rd., 1YSS). Energy of activation \,alue of about 3Y.XkJ.mole- ’ was found by Kunitr (lY52) for yeast pyrophosphatase.
For valid assay of enzymatic activity, it is necessary to ensure that the substrate concentration is not limiting the reaction velocity in the assay procedure. A study of the effect of varying the substrate (PPi) concentration (Fig. 6) showed that the concentration
Pyrophosphatase
of soils
63
(7 CLARION SO
100
0
activity
I
20
,
I
I
I
,
40 60 80 100 120 PPi CONCENTRATION fmMl
6. Effect of substrate
(PPi)
concentration
of Pi in assay of soil pyrophosphatase
140
on release
activity.
adopted (50m~) was satisfactory. At a PPi concentration above 60m~, however, the enzyme reaction velocity decreased. The magnitude of this decrease was greater as the PPi concentration increased. This lower rate of PPr hydrolysis at high concentration seems due to two factors. One is that, at high concentrations, the substrate (free PPi) is known to inhibit pyrophosphatase, and the other is that Mg” is required for activation of PPi for binding with the enzyme (Butler, 1971). The results (Fig. 6) indicate either that, at high PPi concentrations, not enough Mg2 + was present in the soils to produce enough mono-magnesium PPi complex, the substrate for this enzyme, or that PPi inhibited pyrophosphatase. With the exception of one soil (Clarion), the soils used contained similar amounts of exchangeable Mg*+ (Table 1); therefore, the rate-limiting step with increasing the PPi concentration was observed at similar PPi concentration (Fig. 6). Similar curves have been reported by Howard and Lundgren (1970) for pyrophosphatase of F. ,~e~~~o~i~aFls. Michaelis-Menten
constant
Figure 7 shows plots of the three possible linear transformations of the Michaelis-Menten equation
Table 2. K,
Fig. 7. The three possible linear plots of the MichaelisMenten equation. Velocity (u) is expressed as pg Pi released.g-’ soil’ 5 h-r and S is in M. A, Lineweaver-Burk plot (l/v vs l/S); B, Eadie-Hofstee plot (G vs u/S); C, HanesWoolf plot (S/v vs S). n, Okoboji soil; c7, Clarion soil; l, Webster soil.
applied to the pyrophosphatase activity values obtained as a function of PPi concentration. To eliminate the rate-limiting effect of Mg2’ and the inhibition by PPi, *we plotted the activity values obtained with up to 60m~ PPi. The straight lines shown in Fig. 7 are those calculated by regression analysis. As it can be seen from Fig. 7, the results obtained obeyed the three linear transformations of the MichaelisMenten equation.
and F$,.., values of inorganic pyrophosphatase the three transformations of MichaehsMenten
Michaelis-Menten transformation Lineweaver-Burk (l/v vs l/S?
Plot
Soil
K (mM)
Webster Clarion Okoboji Harps
43 51 36 21 38 36 4.5 35 20 35 35 45 34 20 34
Average Eadie-Hofstee (1!vs u/S)
Webster Clarion Okoboji Harps
Plot
Average Hanes-Woolf (Sir vs S)
Webster Clarion Okoboji Harps
Plot
Average *pg Pi released.g-’
soil.5 h-l.
in soils calculated equation
from
I/lndX* 826 730 429 131 529 743 672 422 128 491 730 649 411 130 482
64
w.
A. DICK and ,u, A.
Table 3. Precision
Soil
No. of analyses
Clarion 6 Nicoilet 7 Webster 6 Okoboji 8 Harps 6 Cnnisteo 7 * /tg Pi released.gvariation.
+
Nom Formaldehyde (0.25 ml) Steam sterilization (121 c, 1 h) NaF NazCzO, Na&O,
of method
Pyrophosphatase Mean
Range 33s-337 422432 388-402 220 233 76-X 5 2-60
treatments
activity* SD
CV
1.5 4.5 5.3 6.3 1.6 3.0
0.8 1.1 1.5 1.8 2.1 5.5
335 428 393 227 78 55
’ :ml-S h- ‘. SD, standard
Table 4. Effects of various
Soil treatment
TABATAtlAl
deviation:
CV. coefficient
on pyrophosphatase
Pyrophosphatase activity Nicollet Clarion
activity
of
of soils
of soil specified* Okoboji Harps
335 1’4 17
428 I63 11
227 57 I
78 45 8
24x 701 124
398 321 227
200 129 120
46 4s 4x
* /~g Pi re1eased.g ’ soil.5 h-‘. + A sample of soil (I g) was treated with 0.25 ml of solution made 0.2 M with respect to the inorganic compound specified. After 30 min of equilibration at room temperature (22’C). pyrophosphatase activity was assayed. Na,EDTA. NaCI, N&Cl, KCl, NaNO,, NaNO,: Na*SO,. and toluene (0.25 ml) had no effect,
The K,,, and V,,,:,, values calculated from the plots in Fig. 7 are shown in Table 2. The K,,, values of PPi for pyrophosphatase in four soils ranged from 20 to 50 mM (avg. 35 mM) and the r/;,,‘,, values ranged from 130 to 830 (av SOO) 118 Pi released.g-’ soil 5 h- ‘. The K,,, values of PPi for pyropllosphatase in soils is about one order of magnitude greater than that reported for pyrophosphatase purified from F. ftrrosidn~s (Howard and Lundgren, 1970). This high in apparent K,, value of PPi for pyrophosphatase soils compared with that for the same enzyme of other sources is partly due to sorption of PPi by soil ConstitLIents. The conformity of the reaction rates to the three linear transformations of the Michaelis-Menten equation (Fig. 7). however. suggests that sorption of PPi by soils is proportional to the PPi concentration used.
The pyrophosph~ltase activity values of six soils (Tables 1 and 3) ranged from 55 to 42X (av 253) pg Pi re1eased.g’ soil 5 h ‘. and the standard deviation of the activity determinations ranged from 1.6 to 5.3 (av 3.9). The high precision of the method results largely because extraction and determination of the Pi released is quantitative. and the procedures are simple and readily standardized.
Steam sterilization is known to inactivate soil enzymes, but the results obtained after this treatment
showed some apparent pyrophosphatase activity (Table 4). These apparent activities after steam sterilization, however, are much less than those of the untreated soils. The pyrophosphatase activity after heat treatment could be due to nonspecific chemical hydrolysis of PPi by metal ions. Expressed as a percentage of total pyrophosphatase activities in unsterilized soils, the apparent activities after steam sterilization ranged from 2.6”; for Nicollet soil to 10.3”, for Harps soil. Treatment of soils with formaldehyde, Iluoride. oxalate. and carbonate inhibited pyrophosphatase activity. This inhibition seems to be related to binding of PPi (3utler. of the Mg2+ required for activation 1971). Among the chemical treatments studied, toluene, Na +, K+, NH,‘. Cl-. NO,. NOz. SO:-, and EDTA showed no effect on the activity of this enzyme in soils. ilcXiin~~t~~lget?lcntsJournal Paper J-8690 of the lo%:t Agriculture anti Home Economics Experiment Station, Ames, Iowa. Projects
1868. 2082 and 2112.
REFERENCXS
R. W. and H~SSNER L. R. (1969) Hydrolysis and sorption of ortho. pyro, tripoly and trimetaphosphate in 32 midwestern soils. Proc. Soil Sci. SIC. .AM. 33, 622-625. BUTL~K L. G. (1971) Yeast and other inorganic pyrophosphatases. In 7hr En~~?nes (P. D. Boyer. ed.) Vol. 4. pp. 529-541. Academic Press. New York. BLANCHAK
Pyrophosphatase
DICK W. A. and TABATABAI M. A. (1977) Determination of orthophosphate in aqueous solutions containing labile organic and inorganic phosphorus compounds. J. En1+rotz. ouui. 6, 82-85. Dowx.~
and D. T. Mitchell, Eds.) pp. 475-508. John Wiley, New York. GILLIAMJ. W. and SAMPLEE. C. (1968) Hydrolysis of pyrophosphate in soils: pH and biological effects. Soii Sci. 106, 352- 357. HASHI~~~TO I., HUGHESJ. D., and PIUI.EN0. D. JR. (1969) Reaction of triammonium pyrophosphate with soils and soil minerals. hoc. Soil Sci. Sot. Am. 33, 401405. HOFSTEEH. J. (1952) On the evaluation of the constants G, and K,,, in enzyme reactions. Science 116. 329331. H~~SSNER L. R. and PHILLIPSD. P. (1971) ~rophosphat~ hydrolysis in Aooded soil. Proc. Soil Sri. Sot. Am. 35, 379 ~383. HOWARDA. and LUNUGREXD. G. (1970) Inorganic pyrophosphatase from Ferrobacillus ,ferrooxidans (Thiohcillus ferrooxidms).
Can. J. Biochem.
48, 1302-1307.
activity
of soils
65
HUGHESJ. D. and HASHIMOTOI. (1971) Triammonium pyrophosphate as a source of phosphorus for plants. Proc. Soil. Sci. Sot. Am. 35, 643-647. KILMERV. J. and ALEXANDER L. 7. (1949) Methods of making mechanical analyses of soils. Soii Sci. 48, 15-24. K~NITZ M. (1952) Crystalline inorganic pyrophosphatase isolated from baker’s yeast. J. germ.PhJsiof. 35, 423-450. MERIUSL. J. (1960) A rapid method for the determination of organic carbon in soil. Alzrxlytica ckirn. Acta 22, 120-124. NAGANNAB., RAMANA., VEE~UGOPAL B., and SRIPATHIC. E. (1955) Potato pyrophosphatases, ~i~~c~e~. J. 60, 215-223. SKUJINSJ. J. (1967) Enzymes in soil. In Soil ~joc~e~?~isfr~ (A. D. McLaren and G. H. Peterson. Eds.) Vol. 1, pp. 371-414. Marcel Dekker. New York. SKUJINSJ. J., BRAALL., and MCLAKENA. D. (1962) Characterization of phosphatase in a terrestrial soil sterilized with an electron beam. Enzrmolouia 25, 125-133. SUTTONC. D.. GLJNARYD., and LARSENS. (1966) Pyrophosphate as a source of phosphorus for plants-II. &ydrolysis and initial uptake by a barley crop. Soil Sci, 101; 199-204.
_
-
.
.
TABA~ABAI M. A. and BREMNER J. M. (1970) Arylsulfatase activity of soils. Proc. Soil Sci. Sm. Am. 34, 225-229. TABATABA~M. A. and SINGH B. 8. (1976) Rhodanese activity of soils. Soil Sci. Sot. AWL 1. 40, 381-385.