Published 1994

Chapter 37

Soil Enzymes

M. A. TABATABAI, Iowa State University, Ames, Iowa

Nutrient cycling in soils involves biochemical, chemical, and physiochemi-

cal reactions, with the biochemical processes being mediated by microor-
ganisms, plant roots, and soil animals. It is well known that all biochemical
reactions are catalyzed by enzymes, which are proteins with catalytic prop-
erties owing to their power of specific activation. Enzymes are catalysts,
that is, they are substances that without undergoing permanent alteration
cause chemical reactions to proceed at faster rates. In addition, they are
specific for the types of chemical reactions in which they participate. En-
zyme specificity is often dictated by the nature of the groups attached to the
susceptible bonds. For example, both a-chymotrypsin and trypsin are pro-
teolytic enzymes capable of hydrolyzing certain peptide bonds in protein.
a-Chymotrypsin will hydrolyze peptide bonds in which the carbonyl group
of that bond is supplied by L-tryosin, L-phenylalanine, or L-tryptophan.
Trypsin will hydrolyze peptide bonds in which the carbonyl group of the
peptide bond is supplied by L-arginine or L-Iysine. Peptide bonds con-
taining D-amino acids are not hydrolyzed. As another example of the
specificity of enzymes, maltase hydrolyzes maltose to glucose, whereas
cellubiase hydrolyzes cellubiose to glucose but not vice versa. Differences
between the two substrates seem slight in that maltose is an a-glucoside and
cellubiose is a ~-glucoside.
Enzymes are specific activators in that they combine with their sub-
strates in such a stereospecific fashion that they cause changes in the elec-
tronic configuration around certain susceptible bonds. These bonds then
are more easily changed. Physiochemical measurements indicate that
enzyme-catalyzed reactions have lower activation energy than the uncata-
lyzed counterparts and, therefore, have faster rates of reactions (Browman
& Tabatabai, 1978; Tabatabai & Singh, 1979).
Enzymes are denatured by elevated temperature and extreme pH.
Their physiochemical state and their influence on chemical reactions are
markedly dependent on pH, ionic strength, temperature, and the presence
or absence of inhibitors or activators.

Copyright © 1994 Soil Science Society of America, 677 S. Segoe Rd., Madison, WI 53711,
USA. Methods of Soil Analysis, Part 2. Microbiological and Biochemical Properties-SSSA
Book Series, no. 5.
775
776 TABATABAI

About a century ago, Woods (as cited by Skujins, 1978) presented the
first report on oxidizing enzymes, especially peroxidase, in soils at the
annual meeting of the American Association for the Advancement of Sci-
ence in Columbus, OR. Although the progress in soil enzymology was
extremely slow until 1950, exponential progress has been made in this field
within the past four decades. The history of abiontic soil enzyme research
has been elegantly prepared by Skujins (1978). Therefore, soil may be
looked on as a biological entity, that is, a living tissue (Quastel, 1946) with
complex biochemical reactions. The enzyme activity of soils results from
the activity of accumulated enzymes and from those in proliferating mi-
croorganisms. Accumulated enzymes in soils are regarded as enzymes
present and active in a soil in which no microbial proliferation occurs (Kiss
et aI., 1975). Sources of enzymes in soils are primarily the microbial bio-
mass, although they can also originate from plant and animal residues.
Enzyme activities in soils are derived from free enzymes, such as exoen-
zymes released from living cells, endoenzymes released from disintegrating
cells, and enzymes bound to cell constituents (enzymes present in disinte-
grating cells, in cell fragments, and in viable but nonproliferating cells).
Proliferating microorganisms produce enzymes that are released to the
soil, or that remain within- the multiplying cells. Free enzymes in soils are
adsorbed on organic and mineral constituents or complexed with humic
substances, or both. The amount of free enzymes in the soil solution is
minute compared with that in the adsorbed state. Microbial cells or cell
fragments also may exist in an adsorbed state or in suspension. Therefore,
soil as a system of humus and minerals contains both immobilized enzymes,
stabilized by a three-dimensional network of macromolecules, and oc-
cluded microbial cells. Each of the organic and mineral fractions in soil has
a special influence on enzyme activity (McLaren, 1975). Schematic dia-
grams showing the components of the enzyme activity in soils are presented
by Skujins (1976) and Kiss et al. (1975), and the various theories and
hypotheses proposed to explain the protective influence of soil constituents
on enzyme activity are summarized by Tabatabai and Fu (1992).
Anyone who is concerned with studies of enzymes is concerned with
the general area of catalysis, and anyone who is concerned with catalysis is
most certainly concerned with the velocities of chemical reactions (chemi-
cal kinetics). Therefore, if one is to understand the kinetics of an enzyme
reaction, it is fundamental to understand the meaning of the results ob-
tained for a soil sample. Details of enzyme kinetics are not discussed in this
chapter but may be found in any general biochemistry textbook. Kinetics
of enzyme reactions in heterogeneous systems is discussed by McLaren and
Packer (1970).
At the simplest level, when investigating enzyme reaction in soil, one
must develop a so-called assay for an enzyme. This involves adding a
known amount of soil to a solution containing a known concentration of
substrate species and determining the rate at which the substrate is con-
verted to a product under carefully controlled conditions of temperature,
ENZYMES 777

pH, and ionic strength. Before an adequate assay can be designed, how-
ever, one must know (i) the reaction catalyzed in stoichiometric detail; (ii)
the species, besides the substrate, that must be present (e.g., Mg2+ is
required for the reaction catalyzed by pyrophosphatase); (iii) the kinetic
dependence of the reaction on such required species; (iv) the optimum
conditions of temperature, pH, and ionic strength; and (v) a suitable means
for monitoring substrate disappearance or product appearance.
One of the most difficult problems currently facing soil biologists and
biochemists is the separation of the extracellular enzymatic activities from
those associated with the living organisms. Soil handling has a marked
effect on the results obtained in enzyme studies. Ideally, the chemical and
physical properties should not be altered before enzyme assay. Although
antiseptic agents have been used to retard or minimize microbial growth
during the assay, especially in assays involving long incubation times, it is
essential to evaluate the effect of the antiseptic agent on the enzyme-
catalyzed reaction rate before its use in enzyme assay. Studies by Fran-
kenberger and Johanson (1986) on the use of plasmolytic agents and
antiseptics in soil enzyme assays showed that toluene, ethanol, and Triton
X-100 effectively prevented microbial proliferation during the assay of soil
amidase. Each of these agents had a different effect on the activities of
several enzymes that they studied. Toluene is a plasmolytic agent as well as
an antiseptic and has little effect or only slightly inhibits purified prepara-
tion of acid and alkaline phosphatases, a-glucosidase, and invertase. Tol-
uene severely inhibits catalase and dehydrogenase activities, but enhances
the activities of arylsulfatase and urease (Tabatabai & Bremner, 1970a,
1972a; Frankenberger, & Johanson, 1986). The increase of soil arylsulfa-
tase and urease activities in the presence of toluene suggests that, because
of its plasmolytic character, toluene affects the intercellular enzyme activ-
ities of the measured activities in soils. Methods based on irradiation of soil
samples with high-energy radiation sterilization and use of antibiotics have
also been evaluated, but such methods, like antiseptic methods, alter the
enzyme-reaction rates (Tabatabai & Bremner, 1970a).
Although there are reports on extraction of enzymes from soils (for
review see Tabatabai & Fu, 1992), no soil enzyme has been purified to the
extent of those extracted from microorganisms, plant, and animal tissues.
The extracted activities are usually associated with carbohydrate-enzyme
complexes and often difficult to purify. Therefore, most of the studies on
enzyme activities in soils have been based on measuring the rates of en-
zyme-catalyzed reactions in soil samples. A large number of enzyme-cata-
lyzed reactions in soils have been studied, but only a few assay methods
have been thoroughly evaluated to be considered standard assays. There-
fore, this chapter does not deal with all the enzymes investigated in soils
but does cover those assay methods that have been thoroughly evaluated.
The enzymes covered are those important in C, N, P, and S cycling in soils.
Additional review articles include Skujins (1967,1976,1978), Ladd (1978),
Roberge (1978), Tabatabai and Fu (1992), and Dick and Tabatabai (1992).
778 TABATABAI

37-1 PRINCIPLES

37-1.1 Mechanism of Enzyme Action

The two most remarkable properties of enzymes are their specificity

and catalytic efficiency, and it is in these properties that enzymes differ
most strikingly from simple catalysts. When it is possible to compare the
enzymatic rates with nonenzymatic counterparts, one finds that enzymes
enhance the reaction by several orders of magnitude (Segel, 1975).
Most theories and mathematical analyses of enzyme reactions are
based on the concept that an enzyme acts by forming a complex or com-
pound with t~e substrate. Presumably the complex of enzyme and sub-
strate is unstable and proceeds through a step or number of steps of
rearrangement to form the product plus the original enzyme. This theory
of enzyme action was proposed by Michaelis and Menten (1913) and may
be expressed by the following equation:
kl k3
S+E~ ES~E+P [1]
k2
where S is the substrate, E is the enzyme, ES is the intermediate enzyme-
substrate complex, P is the product of the reaction, and k 1 , k 2 , and k3 are
the respective reaction velocity constants or rate constants of the three
processes.
The reaction of any enzyme is generally observed by measuring the
rate of the chemical reaction being catalyzed by the enzyme. Equations
describing the kinetics of various types of chemical reactions may be found
in physical chemistry textbooks, and rate equations for enzyme reactions
may be found in Reiner (1959), Alberty (1956), and Segel (1975). Equa-
tions describing the kinetics of enzyme reactions in heterogeneous systems
are described by McLaren and Packer (1970).

37-2 FACTORS AFFECTING RATES OF ENZYME REACTIONS

37-2.1 Concentration of Enzyme and Substrate

Most enzyme-catalyzed reactions may be described as either a zero-

order reaction (reaction where the rate is constant and independent of
substrate concentration) or first-order reaction (reaction where the rate at
any time is proportional to the existing substrate concentration). The zero-
order reaction expressed in a differential equation takes the form of dx/
dt = k, upon integration k = x/to The differential equation for the first-
order reaction is dx/dt = k(a -x), and upon integration is

1 a 2.303 a
k=( In a-x = -t- log a-x· [2]
ENZYMES 779

Vmax

[S]
Fig. 37-1. The rectangular hyperbola describing the effect of substrate concentration [S] on
reaction velocity (v).

In the first-order reaction, a is the initial substrate concentration. In both

the zero-order and first-order equation, x is the concentration of converted
substrate, t is the unit of time (usually in minutes), and k is the propor-
tionality constant. Irrespective of the reaction order, the rate of the reac-
tion is proportional to the enzyme concentration. Therefore, the value of
the proportionality constant k may be considered to consist of a combina-
tion of enzyme concentration and a constant. But since the concentration
of the enzyme does not change during the reaction, it may be considered
as part of the proportionality constant.
When the enzyme concentration is held constant and the substrate
concentration is allowed to vary over a wide range, the velocity of the
enzyme-catalyzed reaction may be described by a rectangular hyperbola
(Fig. 37-1). At low substrate concentrations, the velocity of the reaction
follows a first-order reaction equation. As higher concentrations of sub-
strate are reached, a maximum velocity is obtained independent of further
amounts of substrate, and the zero-order reaction equation applies. The
magnitude of the maximum velocity is proportional to the concentration of
the enzyme; the higher the enzyme concentration, the higher the maximum
velocity. A mathematical derivation of a rate equation for the effect of
substrate concentration on the velocity of enzyme-catalyzed reaction was
first introduced by Michaelis and Menten (1913). Derivatives of their equa-
tion are given by Neilands and Stumpf (1964) and White et al. (1968). From
the Michaelis-Menten theory of enzyme action expressed in Eq. [1], it
follows that the reaction rate (v) is, at any moment, equal to k3 ES. When
S ~ 00, the reaction rate approaches a maximum (Vmax) equal to k3 E t ,
780 TABATABAI

where E t = E + ES. If initial reaction rates are measured, the substrate

concentration will remain practically constant during the time of measure-
ment. Further, if S» E t , only a negligible amount of substrate can accu-
mulate in the form of the intermediate complex (ES). From this it follows
that: (i) the concentration of free substrate may be set equal to the total
substrate concentration, and (ii) the rate of disappearance of substrate
equals the rate of formation of product (steady state); when the rate
of formation and disappearance of ES are equal, i.e., when d[ES]/dt
equals - d [ES]/dt, the following equation then describes the steady state:

k1([E] - [ES]) [S] = k 2[ES] + k3[ES]. [3]

Equation [3] may be rearranged to give

[S] ([E]-[ES]) k 2 -k3

[ES] =~ = Km [4]

where the symbols are the same as described before, and [] indicates con-
centration.
The term containing the three velocity constants is usually called K m ,
the Michaelis constant. Numerically, Km is equal to the substrate con-
centration (expressed in moles per liter) at half-maximum velocity rate
(Vmax = 2v). When k2» k3' Km may be set equal to the dissociation
constant (k2/kl) of the enzyme-substrate complex, and lIKm then becomes
the "affinity" constant. If one assumes that such conditions exist in soil
systems, the affinity constants of several enzyme substrates have been
reported (e.g., S20?- and CN- for the rhodanese reaction (Tabatabai &
Singh, 1979).
The Michaelis-Menten equation

[5]
satisfactorily describes the rectangular hyperbola curve (Fig. 37-1), and it
agrees with the theory that enzymes act by forming an unstable complex of
enzyme and substrate. Equation [5] can be written in three linear trans-
formations:

1 1 Km 1
-=--+---
v Vmax Vrnax [S]
Lineweaver-Burk transformation [6]

Hanes-Wolf transformation [7]

ENZYMES 781

v
V = Vrnax - Km . [S]
Eadie-Hofstee transformation. [8]

Plots of the variables of such relationships normally give straight lines.

The values of the slopes and intercepts are commonly used for determi-
nation of the constants from a set of experimental data. Once the Km and
Vmax are known for a particular enzyme reaction under a given set of
conditions, the reaction velocity, V, can be calculated for any substrate
concentration. The Michaelis constant is by far the most fundamental con-
stant in enzyme chemistry. It has the dimensions of concentration (i.e.,
moles per liter), and it is a constant for the enzyme only under rigidly
specified conditions. The Km value is useful in estimating the substrate
concentration necessary to give a maximum velocity. The form of the
Michaelis-Menten equation is such that approximately 10 and 90% of V max
is achieved at substrate concentration corresponding to Km x 10- 1 and
Km x 10, respectively (Neilands & Stumpf, 1964).
Application of the Hanes-Wolf transformation (Eq. [7]) of the
Michaelis-Menten equation in studies of the Km values of soil phosphatases
and arylsulfatase has been reported by Tabatabai and Bremner (1971).
They showed that the apparent Km values of these enzymes are markedly
affected by shaking the soil-substrate mixture during incubation. A similar
conclusion was reached by Tabatabai (1973) in determination of the ap-
parent Km value of urease in soils and soil fractions. Since then, studies on
the Michaelis constants of these enzymes in soils have been reported by
several investigators (Cervelli et aI., 1973; Brams & McLaren, 1974;
Thornton & McLaren, 1975; Pettit et aI., 1976, 1977; Eivazi & Tabatabai,
1977). By changing the buffer and pH used by Tabatabai and Bremner
(1969) in assay of phosphatase activity in soils, Cervelli et aI. (1973) showed
that the substrate, p-nitrophenyl phosphate, is sorbed by soils in the pres-
ence of 0.5 M NaOAc buffer (pH 4.7). They used adsorption parameters
derived from the Freundlich isotherm to determine concentration of the
free substrate and calculate the Km value of acid phosphatase. Irving and
Cosgrove (1976) examined the graphical techniques used in calculation of
the Km values of acid phosphatase and concluded that the linear transfor-
mation of Eadie-Hofstee (Eq. [8]) should be used. Presumably this graph
shows the greatest deviation from the classical Michaelis-Menten equation,
but published work shows that the linear transformations are equally ap-
plicable for estimation of the apparent Km values of enzymes in soils (Pettit
et aI., 1977; Browman & Tabatabai, 1978; Dick & Tabatabai, 1978a; Ta-
batabai & Singh, 1979). Each transformation gives different weight to
errors in the variables (Dowd & Riggs, 1965), and this is reflected in the
variation of estimated Km and V max values derived for any soil enzyme by
using the different plots.
782 TABATABAI

The routine assay of a particular enzyme is usually carried out under

conditions where the enzyme is saturated with the substrate. Under such
conditions, slight changes in the substrate concentration change the mea-
sured reaction rate only slightly. Also, the formation of the product is
occurring at the maximum rate, which usually permits greatest ease in its
determination.
The maximum velocity can be used for determination of enzyme con-
centration if the molecular weight of the enzyme is known. The determi-
nation of molecular weight of soil enzymes is not possible at present,
however, because of the difficulties associated with extraction and purifi-
cation of enzyme proteins in soils (Tabatabai & Fu, 1992). In the soil
system, an assay of enzyme activity is usually employed and expressed in
units such as micrograms or micromoles of product appeared or substrate
disappeared per amount of enzyme taken (e.g., per amount of dry soil or
unit of organic C) per unit of time. Theoretically, measurement of either
the amount of product released or the decrease in concentration of the
substrate is sufficient in enzyme assays, but in practice, measurement of the
product released is usually much more precise because the value is based
on the difference between zero, or close to zero, and finite concentration.
To measure the decrease in substrate concentration after incubation time,
this decrease must be large enough to be determined accurately, and at the
same time, the difference must not be so large to cause a significant change
in the concentration of the substrate. Assays based on measurement of the
decrease in the concentration of the substrate usually are less precise than
those based on measurement of product appearance.
Soil treatments and methods of preparation have a marked effect on
the results obtained in enzyme assays. The effects of air-drying, storage
conditions, storage temperature, and grinding have been documented
(Tabatabai & Bremner, 1970b; Speir & Ross, 1975; Zantua & Bremner,
1975b; Speir, 1977). An increase in activity can usually be obtained by
subdividing the soil into separates and selecting the finer soil fraction for
enzyme assay (A.D. Haig, 1955. Some characteristics of esterase- and
urease-like activity in the soil. Ph.D. diss., Univ. of California, Davis.)
Preparation of a homogenous soil sample is important, because soil en-
zymes are unevenly distributed in the soil fractions (Speir, 1977). These
and other factors (e.g., shaking the soil-substrate mixture) affect enzyme
activities and should be considered in studies involving kinetics of enzyme
reactions in soils.

37-2.2 Temperature

The activity of any chemical reaction increases with temperature, ap-

proximately doubling for every increase of 10 0c. The rate of enzyme-
catalyzed reaction increases as the temperature increases until some high
temperature is reached at which the rate begins to decrease because of
enzyme inactivation. Enzyme-catalyzed reactions, however, are less sen-
sitive to temperature changes than their uncatalyzed counterparts, whereas
ENZYMES 783

the uncatalyzed reaction rate may double with every 10 ac elevation of

temperature, the enzyme-catalyzed reaction rate will increase by a factor
of < 2. The temperature dependence of the rate constant can be described
by the Arrhenius equation:

k = A exp (- Ej RT) [9]

where A is the preexponential factor, Ea is the energy of activation, R is the

gas constants, and T is the temperature in degrees Kelvin. The Arrhenius
equation can be expressed in log form

log k = (- Ej2.303 RT) + log A [10]

where log A and Ea can be determined from the intercept and slope,
respectively, of a linear plot of the dependence of log k on liT at lower
temperatures, generally between 10 to 50 ac.
In studies involving calculation of the activation energies and temper-
ature coefficients (010) of enzyme-catalyzed reactions, it is assumed that
the incubation temperature has no effect on the stability (concentration) of
the enzyme. Experiments to test the validity of this assumption must be
performed at the temperature range desired. The validity of this assump-
tion for soil amidase and pyrophosphatase and plant amidase and urease
activities has been reported (Tabatabai & Dick, 1979; Frankenberger &
Tabatabai, 1981a, 1982). Information on the stability of the enzyme at
various temperatures is essential, because studies of the effect of temper-
ature on enzyme activities in soils have shown that most soil enzymes are
inactivated (denatured) at temperatures between 60 and 70 ac (Halstead,
1964; Tabatabai & Bremner, 1970a; Tabatabai & Singh, 1976; Browman &
Tabatabai, 1978; Tabatabai & Dick, 1979; Frankenberger & Tabatabai,
1980a, 1981a). Usually the stability of enzymes increases as the tempera-
ture decreases below that required for the optimum reaction rate. Al-
though air-drying of field-moist soil may decrease enzyme activities (e.g.,
pyrophosphatase), it seems not to affect the temperature of enzyme inac-
tivation (Tabatabai & Dick, 1979).

37-2.3 pH

Temperature affects enzyme activity in various ways; the number of

factors controlling the effect of pH on an enzyme-catalyzed reaction is even
greater. Changes in H+ concentration influence enzymes, substrates, and
cofactors by altering their ionization and solubility. Variations in such
properties as ionization or solubility influence the rate of catalyzed reac-
tions. Enzymes, being proteinaceous, exhibit marked changes in ionization
from fluctuation in pH. Characteristically, each enzyme has a pH value at
which the rate is optimal, and at each side of this optimum the rate is lower;
thus, the catalytic action of the enzymes operates in a somewhat restricted
pH range. Enzymes are usually most stable in the vicinity of the optimum
784 TABATABAI

pH, and they are irreversibly denatured with extremes in acidity or alka-
linity. It is essential to assay enzyme-catalyzed reactions at, or close to, the
optimum pH of activity. In addition to controlling the ionization of en-
zymes and substrates, the buffer used maintains the optimum pH for the
duration of the reaction; this is especially important in enzyme assays
involving substrates or products with acidic or alkaline properties (e.g.,
formation of NH/ from hydrolysis of amides, urea, L-asparagine, and
L-glutamine in soils). Also, since ionic species of the buffer have a marked
effect on enzyme activity, it is essential that one buffer system be used for
determining the pH profile. The universal buffer has a range from pH 3 to
12 and is suitable for this type of study (Eivazi & Tabatabai, 1977; Juma &
Tabatabai, 1977).
Studies of the effects of pH on the stability of enzymes are important
because extreme changes in pH may irreversibly inactivate the enzymes
that play an essential role in nutrient cycling and humus formation. To test
the extreme changes in pH, the pH stability of urease, acid phosphatase,
alkaline phosphatase, and phosphodiesterase activities of soils was inves-
tigated by Frankenberger and Johanson (1982a) by first incubating a soil
sample at pH values ranging from 1 to 13 for 24 h and then measuring the
activity at its optimum pH under standardized conditions. They reported
that, in general, the decline in enzyme activity in a pH-profile near the
optimum pH range was due to a reversible reaction that involved ionization
or deionization of acidic or basic groups in the active center of the enzyme
protein. Irreversible inactivation of the enzyme was particularly evident at
the lower and higher ranges of acidic and alkaline conditions. Results
reported showed that the pH stability of soil enzymes was highly dependent
on the enzyme and soil being assayed. Frankenberger and Johanson
(1982a) attributed the variation among enzymes and soils to the diversity of
vegetation, microorganisms, and soil fauna as sources contributing to the
enzyme activity and protective sites that allowed entrapment of the enzyme
within colloidal humus and organic-mineral complexes.

37-2.4 Cofactors, Inhibitors, and Ionic Environment

Many enzymes are not catalytically active except when in combination

with a nonprotein component. Generally, these nonprotein cofactors are
heat-stable, dialyzable substances of low molecular weight. Cofactors are
often described as activators, coenzymes, and prosthetic groups, and it is
often difficult to distinguish between the meanings assigned to these terms.
Prosthetic groups are usually defined as substances that are bound firmly
to the enzyme. Coenzymes are usually classified as organic substances that
are freely dissociable from the enzymes. Most coenzymes and prosthetic
groups are changed during the sequence of the catalyzed reaction and need
to be regenerated before they can again participate. Some enzymes are
activated by inorganic ions furnished by mineral salts. Usually these inor-
ganic ions are not changed during the catalyzed process but are required
before the enzyme can carry out the reaction.
ENZYMES 785

Pyrophosphatase is well known for its requirement of inorganic cat-

ions for its activity. Dick and Tabatabai (1983) showed that the inorganic
pyrophosphatase activity in three soils decreased when exchangeable and
soluble metals were removed by leaching with 1 M NH40Ac (pH 8). The
effect of added metal ions at various concentrations in the leached soils
showed that, at certain concentrations, Ba2+, Ca2+, C02 + , Mg2+, Mn2+,
Ni2+, and Zn 2+ promoted, K+ and Na + had no effect, and Fe 2+ and Cr2+
decreased pyrophosphatase activity. At high concentrations (> 50 mM),
C02+, Mn2+, NF+, and Zn 2+ inhibited pyrophosphatase activity in two
soils. The concentration of metal ion needed for optimum activity of py-
rophosphatase varied among the soils. The efficiency of the metal ions at
optimum concentrations (average percentage increase for three soils in
parentheses) in promoting pyrophosphatase was Ca2+ (47) > Mg2+
(42) > Ba2+ = C02+ (29) > NF+ (27) > Zn2+ (20) > Mn 2+ (16). Soil
pyrophosphatase in the presence of 200 mM CaCl2 or MgCl 2 was protected
against inactivation by heat (90°C for up to 30 min).
A large variety of chemical reagents are used to inhibit enzyme reac-
tions. Valuable information might be provided about the essential compo-
nents of complex enzyme systems by using inhibitors. Some inhibitors are
able to react with active groups on the enzyme. Inhibition of enzyme
activity by the inhibitors p-chloromercuribenzoate Hg2+ , and Ag+ might
suggest that SH groups in the protein molecule are essential for activity.
Other reagents might react with activators or coenzymes to inhibit enzyme
activity. For example, chelating agents that combine with certain metals
might illustrate the necessity of a metal in a catalyzed reaction. Further-
more, many reagents inhibit enzyme reactions because they have molecu-
lar features similar to the substrate. Arsenate resembles phosphate and is
able to block many of the reactions in which phosphate takes part. Com-
petitive inhibition of soil arylsulfatase by phosphate, arsenate, tungstate,
and molybdate has been demonstrated (AI-Khafaji & Tabatabai, 1979).
Salts may exert marked influences on enzyme activity. Alberty (1956)
has pointed out that changes in electrolyte concentration may cause
changes in the activity coefficients of the reactants and the activated com-
plex. Also, the degree of ionization of acidic and basic groups in the protein
molecule may be changed by the presence of salts. All these effects may
result in either accelerating or slowing down the catalytic activity.

37-2.5 Enzyme Inhibition

The subject of enzyme inhibition requires special consideration, be-

cause soils receive a variety of organic and inorganic chemicals. Some of
the compounds, such as fertilizers, pesticides, and municipal and industrial
wastes, are added as parts of soil and crop management. Other com-
pounds, such as salts of trace elements, are added to soils as impurities in
lime and fertilizers and as industrial pollutants. Also, inhibition of soil
enzymes for controlling the release of fertilizer has received attention since
the 1970s. Most of this research, however, has been concerned with use of
786 TABATABAI

enzyme inhibitors for controlling urea hydrolysis in soils. Unfortunately,

most of the compounds tested for inhibition of urease in soils are general
enzyme inhibitors, affecting not just urease activity but a wide range of soil
enzymes. Generally, little work has been done on the mechanisms of in-
hibition of soil enzymes by various inhibitors.
An inhibitor may produce an irreversible or reversible decrease in the
enzyme rate constant. An irreversible inhibitor forms a stable compound,
often by the formation of a covalent bond with a particular amino acid
residue in the enzyme structure, which is essential for the catalytic activity.
The phenomenon of reversible inhibition is characterized by an equilib-
rium between the enzyme and inhibitor defined by an equilibrium constant
(Kj ), which is a measure of the affinity of the enzyme for the inhibitor.
Although kinetics of enzyme inhibition is complex and varies with the
enzyme, substrate, and inhibitor, three main groups of reversible inhibition
can be distinguished by their characteristic effects on plots of l/v vs. l/[S]
(Lineweaver-Burk plot). The kinetics of most enzyme reactions in the
presence of varying concentrations of inhibitors are such that the linear
double reciprocal plots of l/v vs. l/[S] show similar lines as in the absence
of inhibitor except that (i) the slope, (ii) the intercept, or (iii) both are
altered. The first case is called competitive, the second uncompetitive, and
the third noncompetitive inhibition. The kinetic diagnostic for uncompet-
itive inhibition (case ii) is that in the double reciprocal plot the slopes
remain constant (i.e., the lines are parallel) but the intercepts vary. This
form of inhibition is rare with single substrates where one possible but not
highly likely mechanism entails combination of the inhibitor exclusively
with the ES complex. This type of mechanism, however, is more common
in more complex cases. The other two cases are more common with single
substrates and are likely mechanisms of inhibition of most soil enzymes.
Competitive inhibition depends on the lack of absolute specificity of
the chemical reactivity on the active site. The enzyme active site combines
more or less loosely with the inhibitor, which is possibly structurally related
to the substrate, thus preventing the attachment of the substrate to the
enzyme-binding site. The inhibitor and substrate, therefore, compete for
the same active site forming enzyme-substrate (ES) and enzyme-inhibitor
(EI) complexes, respectively; ESI complexes are not produced. The extent
of inhibition depends on the relative concentrations of substrate and in-
hibitor in the system. This type of inhibition is indicated by the Line-
weaver-Burk plot (Eq. [6]); a competitive inhibitor increases the slope of
the line without changing the intercept (Fig. 37-2). Therefore, the appar-
ent Km value is higher in the presence than in the absence of inhibitor, but
the V max value remains unchanged. In this type of inhibition, the velocity
of the reaction depends on the inhibitor concentration, the substrate con-
centration, and the relative affinities of the inhibitor and substrate for the
enzyme.
Noncompetitive inhibition cannot be reversed by a simple expedient
of raising the substrate concentration. In this type of inhibition, the sub-
strate combines equally well with the enzyme or its ES complex. In this
ENZYMES 787

Km
Siope o - -
Vmax
tal

Ibl

1
Km
1
(Sl
Fig. 37-2. Lineweaver-Burk plots of the Michaelis-Menten equation describing competitive
inhibition; (a) in the presence of inhibitor and (b) in the absence of inhibitor.

lal
Km
v Slope 0 Vmax

Ibl

Km
1
(S]
Fig. 37-3. Lineweaver-Burk plots of the Michaelis-Menten equation describing noncompeti-
tive inhibition; (a) in the presence of inhibitor and (b) in the absence of inhibitor.

case, three complexes are formed: ES, EI, and ESI complexes, and of
these only the ES complex breaks down to products. This type of inhibition
is not completely overcome by increasing the substrate concentration.
Lineweaver-Burk (Eq. [6]) plots yield lines showing increases in both the
intercept and slope of the line in the presence than in the absence of
inhibitor (Fig. 37-3). Therefore, the apparent Km value is not changed, but
the Vmax is decreased. The amount of inhibition will accordingly depend on
the inhibitor concentration and the affinity of the inhibitor to the enzyme,
and it will not be influenced by the substrate concentration. Noncompeti-
tive inhibitors apparently combine with an enzyme at a point other than
the attachment of the substrate. They are then able to exert an effect on the
active site even though they are situated some distance away. Enzyme
inhibition by metal ions often shows noncompetitive kinetics.
788 TABATABAI

37-2.6 Enzymes in Soils

Soil is a living system where all biochemical activities proceed through

enzymatic processes. Enzymes accumulated in soils are present as free
enzymes, such as exoenzymes released from living cells, endoenzymes re-
leased from disintegrating cells, and enzymes bound to cell constituents
such as disintegrating cells, in cell fragments, and in viable but nonprolif-
erating cells (Kiss et aI., 1975). Most of the enzymes added to soils by
decaying microbial tissues and plant and animal residues are likely de-
graded by soil proteases, and what remains is incorporated with the humus.
Soil, therefore, can be viewed as a system of humus and minerals contain-
ing both immobilized enzymes and occluded microbial cells (McLaren,
1975).
Many of the fundamental questions concerning the origin, location,
and persistence of soil enzymes remain unanswered. Although most or-
ganic materials are metabolized rapidly by microorganisms, both in vitro
and in vivo, many enzyme-proteins persist in soils as active moieties for a
long time (Skujins & McLaren, 1968). For example, experiments have
shown that addition of urease to soil resulted in temporary increases in
urease activity, suggesting that the added urease is either inactivated or
destroyed by soil protease (Conrad, 1940c; Stojanovic, 1959; Roberge,
1970; Burns et aI., 1972; Zantua & Bremner, 1976). The existence of
mechanisms to protect enzymes in soils is supported by work showing that
soil enzyme activity is independent of the microbial population (e.g., Paul-
son & Kurtz, 1969; Ramirez-Martinez & McLaren, 1966b). Also, it has
been shown that urease activity could be detected in soils stored for de-
cades and that this activity is better correlated with organic C than with
microbial numbers (Skujins & McLaren, 1968, 1969). It has been suggested
that native soil urease resides in organic colloidal particles with pores large
enough for water, urea, NH3 , and CO 2 to pass freely, but the pores are
small enough to exclude such enzyme as pronase (Burns et aI., 1972); the
added but not the native urease is destroyed by pronase. A model describ-
ing this system and the possible mechanisms involved in binding enzymes
by soil organic matter are discussed by Paul and McLaren (1975). Recent
work by Lai and Tabatabai (1992) showed that the kinetic and thermo-
dynamic parameters of jackbean urease were significantly altered by ad-
sorption on clay or soil mineral constituents.
Among the many factors that may affect enzyme activities in soils,
cropping history, soil amendments, and some environmental factors have
a substantial influence. For example, Stojanovic (1959) found marked sea-
sonal variation in urease activity in Mississippi soils. Similarly, Cooper
(1972) observed seasonal variation in arylsulfatase activity in Nigerian
soils. He reported that arylsulfatase activity decreased during dry periods,
increased after rainfall, and reached a maximum toward the end of the
rainy season. The cycle was repeated during the dry seasons. Waterlogging
of soils produces changes in Eh and pH, and consequently in the activity of
ENZYMES 789

many enzymes (Pulford & Tabatabai, 1988). Other studies on the extra-
cellular enzyme activities in ecosystems have shown that vegetation, agri-
cultural chemicals, and industrial pollutants have a marked influence on
soil enzymes. Generally, the effects observed differ markedly and depend
on many factors, including the soil type, dose of the chemical, and condi-
tions of the study. Because of the importance of urea as a fertilizer in world
agriculture and the potential of soil urease to cause volatilization of NH3
produced from urea hydrolysis in soils, several workers have investigated
various enzyme inhibitors for agricu~ural application. A detailed discus-
sion on the inhibitors tested on urease activity in soils and enzyme in-
teractions with various agricultural and industrial chemicals, including pes-
ticides, is presented by Kiss et al. (1975) and Bremner and Mulvaney
(1978).
Although the patterns of distribution of several enzymes in soil profiles
have been studied by many workers, the relative factors that affect the
activity of enzymes through the profile have not been clearly established.
Like other biochemical reactions in soils, however, enzyme activities are
associated with organic matter distribution profile and generally decrease
with depth. This has been demonstsated for arylsulfatase (Tabatabai &
Bremner, 1970b), urease (Tabatabal, 1977), inorganic pyrophosphatase
(Tabatabai & Dick, 1979), amidase (Frankenberger & Tabatabai, 1981a),
L-asparaginase (Frankenberger & Tabatabai, 1991b), and L-glutaminase
(Frankenberger & Tabatabai, 1991d).
Several methods have been proposed for soil sterilization or inhibition
of microbial proliferation that allow assay of enzyme activities (Franken-
berger & Johanson, 1986). An ideal sterilization agent for extracellular
enzyme detection in soil would be one that would completely inhibit all
microbial activities, but not lyse the cells and not affect the extracellular
enzymes. Unfortunately, however, no such universal agent is yet available.
Toluene has been the most widely used microbial inhibitor, but its
usefulness is limited to the assay procedures that involve a few hours of
incubation. Assay procedures involving long times of incubation with or
without toluene should be avoided, because the risk involved from micro-
bial proliferation increases as the time of incubation increases. It is be-
lieved that in assay procedures involving short incubation times, toluene
inhibits the synthesis of enzymes by living cells and prevents assimilation of
the reaction products. Toluene has also shown to be a plasmolytic agent in
certain groups of microorganisms, in which it apparently induces the re-
lease of intracellular enzymes (Skujins, 1967). A critical examination of the
effect of toluene on soil microorganisms has been made by Beck and
Poschenrieder (1963). They showed that the inhibitory effect and concen-
tration of toluene needed to suppress microbial activity are strikingly de-
pendent on the pretreatment and moisture content of a particular soil. To
suppress microbial proliferation in an air-dried, naturally moist, or dried
and remoistened soil, at least 20% toluene is necessary. In a soil suspen-
sion, however, 5 to 10% toluene is sufficient. Gram-positive bacteria and
790 TABATABAI

Streptomyces are considerably more resistant to toluene treatment than are

gram-negative bacteria. In addition to the effect of toluene, the effect of
dimethyl sulfoxide, ethanol, and Triton X-lOO on soil enzyme activities was
evaluated by Frankenberger and Johanson (1986). They reported that tol-
uene, a plasmolytic agent as well as an antiseptic, had little effect or only
slightly inhibited purified preparations of acid and alkaline phosphatases,
a-glucosidase, and invertase, but severely inhibited catalase and dehydro-
genase activities. The soil enzymes, arylsulfatase and urease, were en-
hanced (1.30- to 1.34-fold) in the presence of toluene, suggesting that its
plasmolytic character was affecting the intercellular enzyme contribution of
the measured activities in soils.
Irradiation of soil with high-energy radiation is another method used
for soil sterilization. Dunn et al. (1948) introduced the electron beam for
heatless sterilization of soil. McLaren et al. (1957) showed that soil can be
sterilized by an electron beam of sufficient energy and intensity. They
found that 2 x 106 roentgen-equivalent-physical (rep) dose was necessary
for a 1-g sample; urease activity was retained in the sterilized soil. The
effects of ionizing radiation on soil constituents have been reviewed by
Skujins (1967), McLaren (1969), and Cawse (1975). Generally, microor-
ganisms and enzymes are killed or inactivated as an exponential function of
dose of radiation, but the dose required depends on the soil type, soil
moisture, and genus of the organism.

37-3 ASSAY OF ENZYMES IN SOILS

37-3.1 Amidobydrolases (L-Asparaginase, L·Glutaminase,

Amidase, and Urease)

37-3.1.1 Introduction
Several amidohydrolases are present in soils. All are involved in hy-
drolysis of native and added organic N to soils. Among these, L-asparagi-
nase, L-glutaminase, amidase, and urease are the most important.
The enzyme L-asparaginase (L-asparagine amidohydrolase EC
3.5.1.1) plays an important role in N mineralization of soils. The chemical
nature of N in soils is such that a large proportion (15-25 % ) of the total soil
N is often released as NH/ by acid hydrolysis (6 N of HCI). Some evidence
suggests that a portion of the released NH/ comes from the hydrolysis of
amide (asparagine and glutamine) residues in soil organic matter (Sowden,
1958). Bremner (1955) reported that after acid hydrolysis of humic prep-
arations, 7.3 to 12.6% of the total N was in the form of amide-No More-
over, Sowden (1958) reported that a percentage of the NH/ released
during acid hydrolysis was equal to or nearly equal to the sum of aspartic
acid-N plus glutamic acid-N derived from asparagine and glutamine.
ENZYMES 791

L-Asparaginase activity was first detected in soils by Drobni'k (1956).

This enzyme catalyzes the hydrolysis of L-asparagine, producing L-aspartic
acid and NH3 as shown below:

NH2 NH2
I L-asparaginase I
H-C-COOH + H 20 .. H-C-COOH + NH3 [11]
I I
CH2 CH2
I I
CO COOH
I
NH2

L-Asparaginase isolated from guinea pig serum specifically acts only on the
L-isomer form of asparagine and not the D-isomer (Tower et al., 1963).
However, the stereospecificity of L-asparaginase derived from Alcaligenes
eutrophus is such that both D-asparagine and L-glutamine have been re-
ported as substrates (Allison et al., 1971). This enzyme is widely distrib-
uted in nature. It has been detected in both plants and microorganisms
(Wriston, 1971).
Asparaginases have been shown to vary widely in different strains of
microorganisms. Campbell et al. (1967) found two asparaginases in Es-
cherichia coli B and designated them EC-1 and EC-2. These two enzymes
differ in solubility, chromatographic behavior, antilymphoma activity, and
optimum pH. Also, E. coli K-12 contains two asparaginases but only one
appears in cells grown under anaerobic conditions (Cedar & Schwartz,
1967). Cells of Pseudomonas fiuorescens contain two inducible isoenzymes
of asparaginase (Eremenko et al., 1975), one that hydrolyzes only L-
asparagine (asparaginase A) and one that hydrolyzes L-asparagine, L-
glutamine, and D-asparagine (asparaginase AG).
Soils have been tested for L-asparaginase activity by Beck and Po-
schenrieder (1963), simply by adding L-asparagine to soils and monitoring
the NH/ released, but a standard assay procedure was not developed
using systematic studies of factors affecting the release of NH/ . The pro-
posed method by Frankenberger and Tabatabai (1991a) involves determi-
nation of the NH/ -N released by L-asparaginase activity when soil is
incubated with buffered (0.1 M of THAM, pH 10) L-asparagine solution
and toluene at 37°C.
L-Glutaminase is among the amidohydrolases that play an important
role in supplying N to plants. This hydrolase is specific and acts on C-N
bonds other than peptide bonds in linear amides.
L-Glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) activity in
soils was first detected by Galstyan and Saakyan (1973). The reaction
catalyzed by this enzyme involves the hydrolysis of L-glutamine yielding
L-glutamic acid and NH3 as shown:
792 TABATABAI

COOH COOH
I L-glutaminase I
NH2-C-H + H 20 .. NH2-C-H + NH3 [12]
I I
CH2 CH2
I I
CH2 CH2
I I
CO COOH
I
NH2

L-Glutaminase is widely distributed in nature. It has been detected in

several animals (Sayre & Roberts, 1958), plants (Bidwell, 1974), and mi-
croorganisms (Imada et aI., 1973). Microorganisms that have shown to
contain L-glutaminase activity include bacteria, yeasts, and fungi.
Plants and microorganisms are probable sources of L-glutaminase ac-
tivity in soils. However, the main source is believed to be microbial in
nature. Among the bacteria, very high levels of L-glutaminase activity have
been reported in Achromobacteraceae soil isolates (Roberts et aI., 1972).
Fungal species that are known to produce L-glutaminase include Tilach-
lidium humicola, Verticillium malthousei, and Penicillium urticae (Imada et
aI., 1973). The proposed method by Frankenberger and Tabatabai (1991c)
involves determination of the NH/ -N released by L-glutaminase activity
when soil is incubated with buffered (0.1 M of TRAM, pH 10) L-glutamine
solution and toluene at 37°C,
Amidase (acylamide amidohydrolase, EC 3.5.1.4) is the enzyme that
catalyzes the hydrolysis of ami des and procedures NH3 and the correspond-
ing carboxylic acid:

RCONH2 + H 20 ~ NH3 + R·COOH. [13]

Amidase acts on C-N bonds other than peptide bonds in linear

amides. It is specific for aliphatic and aryl amides cannot act as substrates
(Kelly & Clarke, 1962; Florkin & Stotz, 1964). This enzyme is widely
distributed in nature. It has been detected in animals and microorganisms
(Bray et aI., 1949; Clarke, 1970). Amidase is present in leaves of corn (Zea
mays L.), sorghum (Sorghum bicolor L. Moench), alfalfa (Medicago sativa
L.), and soybean (Glycine max L.) (Frankenberger & Tabatabai, 1982).
Microorganisms shown to possess amidase activity include bacteria
(Clarke, 1970; Frankenberger & Tabatabai, 1985), yeast (Joshi & Handler,
1962), and fungi (Hynes, 1970, 1975). The substrates of this enzyme are
sources of N for plants (Cantarella & Tabatabai, 1983). The method pro-
posed involves determination of the NH4-N released by amidase activity
when soil is incubated with buffered (0.1 M of THAM, pH 8.5) amide
solution and toluene at 37°C (Frankenberger & Tabatabai, 1980a,b;
1981a,b).
ENZYMES 793

Urease (urea amidohydolase, EC 3.5.1.5) is the enzyme that catalyzes

the hydrolysis of urea to CO2 and NH3:

[14]

It acts on C-N bonds other than peptide bonds in linear ami des and thus
belongs to a group of enzymes that include glutaminase and amidase. Since
two C-N bonds are broken in hydrolysis of urea by urease, it is evident that
the stoichiometric relation in the equation is the result of component re-
actions. Several studies have been conducted to determine the mechanism
of urease action, and the work by Gorin (1959) and Blakeley et al. (1969)
has provided convincing evidence that carbamate is the intermediate in a
two-step reaction. This reaction is summarized by Reithel (1971) as fol-
lows:

OH ONH+
/ 4
/
O=C +NH3 :;;:::: O=C [15]

""NH2 ""NH2 [12]

Evidence derived from kinetic data suggests that urease forms a carbamoyl
complex:

H2N-C-enz
II
o
as one of the ES complexes, and presumably water is the acceptor in a
carbamoyl transfer reaction. Therefore, carbamate is the obligatory sub-
strate for the second step in the overall reaction (Reithel, 1971). Since the
proposed mechanism is based on kinetic studies, direct evidence for this
mechanism is still needed.
Urease is very widely distributed in nature. It has been detected in
microorganisms, plants, and animals. Its presence in soils was first reported
by Rotini (1935). Studies by Conrad (1940a,b,c; 1942a,b; 1943) provided
the basic information about this enzyme in soil systems. Several reasons
could account for the large amount of literature on urease. These include:
1. Urease was the first enzyme protein to be crystallized in 1926 by
Sumner (1951).
2. Its reaction products (C0 2 and NH3) are relatively easy to deter-
mine.
3. It can be purified from various sources.
4. It can give a model for a simple enzyme-catalyzed reaction.
5. It is available commercially at reasonable prices.
6. Urea is an important N fertilizer.
794 TABATABAI

Besides the several reviews available on urease in the biochemistry liter-

ature (Sumner, 1951; Varner, 1960; Reithel, 1971), there are numerous
articles covering some aspects of soil urease (Skujins, 1967, 1976; Kupre-
vich & Shcherbakova, 1971; Sequi, 1974; Kiss et aI., 1975; Bremner &
Mulvaney, 1978).
Soil urease activity apparently is optimum at tris(hydroxymethyl) ami-
nome thane (THAM, Fisher Scientific Co., Chicago, or TRIS) buffer pH of
8.5 to 9.0, which is about 2 pH units greater than that for optimal urease
in solution (Tabatabai & Bremner, 1972a); the Km values of soil urease for
urea range from 1.1 to 3.4 mM (Tabatabai, 1973).
A variety of methods has been used for assay of urease activity in soils
(Tabatabai & Bremner, 1972a; Bremner & Mulvaney, 1978). Most of these
involve determination of the NH/ released on incubation of toluene-
treated soil with buffered urea solution, but assays have also been per-
formed by estimation of the CO2 released or urea hydrolyzed on incubation
(Conrad, 1940c; Porter, 1965; Skujins & McLaren, 1969; Douglas & Brem-
ner, 1971). Several of the methods adopted have not involved use of a
buffer to control pH (Porter, 1965; Douglas & Bremner, 1971) or addition
of toluene to inhibit microbial proliferation (McLaren et aI., 1957; Porter,
1965; Paulson & Kurtz, 1969).
The method proposed by Tabatabai and Bremner (1972a) involving
estimation of the NH/ released has been thoroughly evaluated. This
method involves use of toluene, and it has been shown to be applicable to
soils that fix NH/ and is described here.
The other method described involves estimation of the rate of urea
hydrolysis in soils by determination of the urea remaining after incubation
of soil with a urea solution. The difference between the amount of urea
added and that recovered after incubation for a specific time is taken as
an estimate of urease activity. Urea may be estimated by a colorimetric
method involving the reaction of urea with p-dimethyl-aminobenzaldehyde
(Porter, 1965) or diacetlymonoxime (DAM) and thiosemicarbazide (TSC)
(Douglas & Bremner, 1970a). Colorimetric determination of urea by
DAM and TSC, however, is more sensitive than by p-dimethylaminoben-
zaldehyde. These methods do not give the actual urease activity in soils,
because neither involves use of a buffer to control the pH of the incubation
mixture (for the problems associated with such methods, see Burns, 1978),
however, they are useful for estimation of the degree of urea hydrolysis in
soils. The method based on determination of urea with DAM and TSC is
described.

37-3.1.2 Principles
The methods, designed for assay of L-asparaginase, L-glutaminase,
amidase, or urease under optimum conditions, are based on determination
of the NH/ released when soil is incubated with THAM buffer, L-aspar-
agine, L-glutamine, formamide, or urea solution, and toluene at 37°C for
2 h. The NH/ released is determined by treatment of the incubated soil
ENZYMES 795

sample with 2 M of KCl containing Ag2S04 or uranyl acetate (to stop

enzyme activity) and steam distillation of an aliquot of the resulting soil
suspension with MgO for 4 min. In these methods the enzymes are assayed
at optimum buffer pH and substrate concentration.
The second method described for urease assay is designed for estima-
tion of the rate of urea hydrolysis in soils. It involves colorimetric deter-
mination of the urea remaining after incubation at 37°C for 5 h of soil with
urea solution. The amount of urea hydrolyzed g-l of soilS h- 1 is estimated
from the difference between the initial amount of urea added and that
recovered after incubation.

37-3.1.3 Assay Methods

37-3.1.3.1 L-Asparaginase (Frankenberger & Tabatabai, 1991a,b)

37-3.1.3.1.1 Special Apparatus

1. Volumetric flasks, 50 mL.
2. Incubator, ordinary incubator, or temperature-controlled water
bath.
3. Steam distillation apparatus (see chapter 40 in this book).

37-3.1.3.1.2 Reagents
1. Toluene, Fisher certified reagent (Fisher Scientific Co., Chicago).
2. Tris(hydroxymethyl)aminomethane (TRAM) buffer 0.1 M, pH 10:
Dissolve 12.2 g of THAM (Fisher certified reagent, Fisher Scien-
tific Co., Chicago) in about 700 mL of water, titrate the pH of the
solution to 10 by addition of approximately 0.1 M NaOH, and
dilute with water to 1 L.
3. L-Asparagine solution, 0.5 M: Dissolve 1.65 g of L-asparagine
(Sigma Chemical Co., St. Louis, MO) in about 20 mL of tris(hy-
droxymethyl)aminomethane (THAM) buffer, and dilute the solu-
tion to 25 mL with THAM buffer. Mix the contents while running
hot tap water over the flasks.
4. Potassium chloride (2.5 M)-silver sulfate (100 ppm) (KCI-Ag2 S0 4 )
solution: Dissolve 100 mg of reagent-grade Ag2S04 in about 700
mL of water, dissolve 188 g of reagent-grade KCI in this solution,
and dilute the solution to 1 L with water.
5. Reagents for determination of NH/-N (MgO, H 3B0 3 -indicator
solution, 0.0025 M H 2 S04 ): Prepare as described in chapter 40.

37-3.1.3.1.3 Procedure
Place 5 g of soil « 2 mm) in a 50-mL volumetric flask, add 0.2 mL of
toluene and 9 mL of THAM buffer, swirl the flask for a few seconds to mix
the contents, add 1 mL of 0.5 M L-asparagine solution, and swirl the flask
again for a few seconds. Then stopper the flask, and place it in an incubator
at 37°C. After 2 h, remove the stopper, add approximately 35 mL of
7% TABATABAI

KCI-Ag2S04 solution, swirl the flask for a few seconds, and allow the flask
to stand until the contents have cooled to room temperature (about 5 min).
Make the contents to 50 mL by addition of KCI-Ag2S04 solution, stopper
the flask, and invert it several times to mix the contents. To determine
NH/ -N in the resulting soil suspension, pipette a 20-mL aliquot of the
suspension into a lOO-mL distillation flask, and determine the NH/-N
released by steam distillation of this aliquot with 0.2 g of MgO for 4 min
(chapter 40).
Controls should be performed in each series of analyses to allow for
NH/-N not derived from L-asparagine through L-asparaginase activity.
To perform controls, follow the procedure described for assay of L-aspar-
aginase activity, but make the addition of 1 mL of 0.5 M L-asparagine
solution after the addition of 35 mL of KCI-Ag2S04 solution.
37-3.1.3.2 L-Glutaminase (Frankenberger & Tabatabai, 1991c,d)

37-3.1.3.2.1 Special Apparatus

As described in section 37-3.1.3.1.1.

37-3.1.3.2.2 Reagents
1. Toluene, Fisher certified reagent (Fisher Scientific Co., Chicago).
2. THAM buffer (0.1 M, pH lO) and KCI-Ag2S04 mixture: prepare as
described in section 37-3.1.3.1.2.
3. L-Glutamine solution, 0.5 M: Dissolve 1.82 g of L-glutamine
(Sigma Chemical Co., St. Louis, MO) in about 20 mL of tris(hy-
droxymethyl)aminomethane (THAM) buffer (0.1 M, pH lO) and
dilute the solution to 25 mL with THAM buffer. Mix the contents
while running hot tap water over the flask.
4. Reagents from determination of NH/-N (MgO, H 3B03 -indicator
solution, 0.0025 M H 2S04): Prepare as described in chapter 40.

37-3.1.3.2.3 Procedure
Follow the procedure described for L-asparaginase activity but use 1
mL of 0.5 M L-glutamine solution instead of 1 mL of 0.5 M L-asparagine
solution.
Controls should be performed in each series of analyses to allow for
NH/ not derived from L-glutamine through L-glutaminase activity. To
perform controls, follow the procedure described for performing controls
in assay of L-asparaginase activity but use steam sterilized soil and 1 mL of
0.5 M L-glutamine instead of 1 mL of 0.5 M L-asparagine.
37-3.1.3.3 Amidase (Frankenberger & Tabatabai, 1980a,b)

37-3.1.3.3.1 Special Apparatus

As described in section 37-3.1.3.1.1.
ENZYMES 797

37-3.1.3.3.2 Reagents
1. Toluene, Fisher certified reagent (Fisher Scientific Co., Chicago).
2. THAM buffer (0.1 M, pH 8.5): Prepare as described in section
37-3.1.3.1.2.
3. Formamide solution (0.50 M): Add 2.0 mL of formamide (Aldrich
certified) into a 100-mL volumetric flask. Make up volume by add-
ing THAM buffer, and mix the contents. Store the solution in a
refrigerator.
4. Potassium chloride (2.5 m)-uranyl acetate (0.005 M) solution: Dis-
solve 2.12 g of reagent-grade UOiC2 H 30 2) 2H 20 in about 700 mL
of water, dissolve 188 g of reagent-grade KCI in this solution, and
dilute the solution to 1 L with water.
5. Reagents for determination of NH/ -N (MgO, H 3B0 3-indicator
solution, 0.0025 M H 2 S04 ): Prepare as described in chapter 40.

37-3.1.3.3.3 Procedure
Follow the procedure described in section 37-3.1.3.1.3 for assay of
L-asparaginase activity but use 1 mL of 0.5 M formamide solution instead
of 1 mL of 0.5 M L-asparagine solution.
Controls should be performed in each series of analyses to allow for
NH/ -N not derived from formamide through amidase activity. To perform
controls, follow the procedure described in section 37-3.1.3.1.3 for per-
forming controls in assay of L-asparaginase activity but use 1 mL of 0.5 M
formamide instead of 1 mL of 0.5 M L-asparagine.

37-3.1.3.4 Urease (Tabatabai & Bremner, 1972a)

37-3.1.3.4.1 Special Apparatus

As described in section 37-3.1.3.1.1.

37-3.1.3.4.2 Reagent
1. Toluene, Fisher certified reagent (Fisher Scientific Co. , Chicago).
2. THAM buffer (0.05 M, pH 9.0) and KCI-Ag2 S0 4 mixture: Prepare
as described in section 37-3.1.3.1.2, but use 0.2 M H 2S0 4 instead
of NaOH to titrate the THAM buffer.
3. Urea solution, 0.2 M: Dissolve 1.2 g of urea (Fisher certified re-
agent, Fisher Scientific Co., Chicago) in about 80 mL of THAM
buffer, and dilute the solution to 100 mL with THAM buffer. Store
the solution in a refrigerator.
4. Potassium chloride (2.5 M)-silver sulfate (100 ppm) (KCI-Ag 2S0 4 )
solution: Prepare as described in section 37-3.1.3.1.2.
5. Reagents for determination of NH/ -N (MgO, H 3BO r indicator
solution, 0.0025 M H 2S0 4 ): Prepare as described in chapter 40.
798 TABATABAI

37-3.1.3.4.3 Procedure
Follow the procedure described in section 37-3.1.3.1.3 for assay of
L-asparaginase activity but use 1 mL of 0.2 M area solution instead of 1 mL
of 0.5 M L-asparagine solution.
Controls should be performed in each series of analyses to allow for
NH/-N not derived from urea through urease activity. To perform con-
trols, follow the procedure described in section 37-3.1.3.1.3 for performing
controls in assay of L-asparaginase activity but use 1 mL of 0.2 M urea
solution instead of 0.5 M of L-asparagine solution.
37-3.1.3.5 Urease by Determination of Urea Remaining!

37-3.1.3.5.1 Special Apparatus

1. Volumetric flasks, 50 mL with glass stoppers.
2. Incubator, ordinary incubator, or temperature-controlled water
bath.
3. Water bath (boiling water).
4. Suction funnels (polyethylene) and filtering funnel stand. (Soil-
moisture Equipment Co., Santa Barbara, CA).
5. Klett-Summerson photoelectric colorimeter or spectrophotometer.

37-3.1.3.5.2 Reagents
1. Urea (substrate) solution: Dissolve 2.0 g of urea (Fisher certified
reagent, Fisher Scientific Co., Chicago) in about 700 mL of water,
and adjust the volume to 1 L with water. This solution contains 2
mg of urealmL. Store this solution in a refrigerator.
2. Phenylmercuric acetate (PMA) solution: Dissolve 50 mg of PMA
(Eastman Organic Chemicals, Rochester, NY) in 1 L of water.
3. Potassium chloride-phenylmercuric acetate (2 M KCI-PMA) solu-
tion: Dissolve 1500 g of reagent-grade KCI in 9 L of water, and add
1 L of PMA solution.
4. Diacetylmonoxime (DAM) solution: Dissolve 2.5 g of DAM
(Fisher Scientific Co., Chicago) in 100 mL of water.
5. Thiosemicarbazide (TSC) solution: Dissolve 0.25 g of TSC (East-
man Organic Chemicals, Rochester, NY) in 100 mL of water.
6. Acid reagent: Add 300 mL of 85% phosphoric acid (H3P0 4 ) and 10
mL of concentrated sulfuric acid (H2S04 ) to 100 mL of water, and
make up the volume to 500 mL with water.
7. Color reagent: Prepare this reagent immediately before use by
adding 25 mL of DAM solution and 10 mL of TSC solution to 500
mL of acid reagent.
8. Standard urea stock solution for standard curve: Dissolve 0.500 g of
urea in about 1500 mL of 2 M potassium chloride-phenylmercuric

lModified from Douglas and Bremner (1970a,b) and Zantua and Bremner (1975a).
ENZYMES 799

acetate (KCI-PMA) solution, and dilute to 2 L with this same so-

lution. If pure, dry urea is used, this solution will contain 250 f.tg of
urea/mL. Store this solution in a refrigerator.

37-3.1.3.5.3 Procedure
Place 5 g of soil « 2 mm, on oven-dry basis) in a 65-mL (2-oz.) glass
bottle, and treat it with 5 mL of urea solution (10 mg of urea). Stopper the
bottle, and incubate at 37°C. After 5 h, remove the stopper, and add 50
mL of 2 M KCI-PMA solution (reagent 3). Stopper the bottle again, and
shake it for 1 h. Filter the soil suspension, under suction, through Whatman
no. 42 filter paper by using a suction funnel and filtering funnel stand.
To determine the urea remaining, pipette an aliquot (1-2 mL) of the
extract containing up to 200 f.tg of urea into a 50-mL volumetric flask, make
the volume to 10 mL with 2 M KCI-PMA solution, and add 30 mL of the
color reagent. Swirl the flask for a few seconds to mix the contents, and
place it in a bath of boiling water. After 30 min, remove the flask from the
water bath, cool it immediately in running water for about 15 min (this can
be accomplished by placing the flask in a deep tray containing cold water,
12-20 0c). Then make the volume to 50 mL with water, and mix thor-
oughly. Measure the intensity of the red color produced with a Klett-
Summerson photoelectric colorimeter fitted with a green (no. 54) filter.
Calculate the urea content of the extract analyzed by reference to a
calibration graph plotted from the results obtained with standards contain-
ing 0, 25, 50, 100, 150, and 200 f.tg of urea. To prepare this graph, dilute 10
mL of the standard urea stock solution to 100 mL with 2 M KCI-PMA
solution in a volumetric flask, and mix thoroughly. Then pipette 0,1,2,4,
6, and 8 mL of aliquots of this working standard solution into 50-mL
volumetric flasks, adjust volumes to 10 mL by adding 2 M KCI-PMA
solution, and proceed as described for urea analysis of the soil extract.
To calculate the amount of urea hydrolyzed in soil during incubation,
divide the amount of total urea recovered by 5 (micrograms of urea re-
covered per gram of soil) and subtract this value from 2000 (micrograms of
urea initially added per gram of soil).

37-3.1.4 Comments
In the assay methods based on determination of the NH/ released,
the buffer described should be used, because other buffers (e.g., phos-
phate, acetate, and universal buffers) give lower results for L-asparaginase,
L-glutaminase, amidase, and urease activities in soils. Of the buffers tested
by Tabatabai and Bremner (1972a), the buffer described prevent NH/
fixation by soils (this has been confirmed by quantitative recovery of NH/
added to Montana vermiculite). The THAM buffer should be prepared by
using H 2S04 , because work by Wall and Laidler (1953) showed that buffers
prepared by treatment of THAM solutions with HCI have an activation
effect on hydrolysis of urea by jackbean (Canavalia ensiformis L.) urease.
800 TABATABAI

The KCI-Ag2S04 solution must be prepared by the addition of KCI to

Ag2S0 4 solution as specified (Ag2S0 4 will not dissolve in KCI solution),
and the soil suspension analyzed for NH/ must be mixed thoroughly
immediately before sampling. The KCI-Ag2S04 treatment used terminates
the enzyme activity, and no additional NH/ release is expected if the soil
suspension is allowed to stand for 2 h before NH/ analysis. If the soil
suspension cannot be analyzed within 4 h after the addition of the KCl-
Ag2S04 reagent, the flask should be stored in a refrigerator.
The KCI-U02(C2H302h·2H20 reagent should be stirred continuously
before use; if this solution is allowed to stand without stirring, KCI will
precipitate slowly out of the solution with time. The purpose of adding the
KCl-uranyl acetate solution to the soil sample after incubation in assay of
amidase activity is to inactivate amidase and allow quantitative determi-
nation of the NH/ released (Jakoby & Fredericks, 1964; Tabatabai &
Bremner, 1972a). Potassium chloride-Ag2S04 can be substituted for KCI-
uranyl acetate provided the NH/ release is determined immediately, be-
cause KCI-Ag2S04 does not completely inactivate amidase activity in soils.
In addition to formamide, acetamide and propionamide may be used as
substrates, but with lower specificity. Assay of amidase in the absence of
toluene indicated that its substrates may induce the synthesis of this en-
zyme by soil microorganisms (Frankenberger & Tabatabai, 1980a).
For a valid assay of enzyme activity, it is necessary to ensure that the
enzyme substrate concentration is not a limiting factor in the assay proce-
dure. The substrate concentration recommended (0.05 M L-asparagine,
L-glutamine, and formamide, and 0.02 M of urea) have been shown to be
satisfactory for assay of amidohydrolase activities in a variety of soils. The
D-isomers of asparagine and glutamine are also hydrolyzed in soils, but at
about 16 and 7% of the L-isomers, respectively, at saturating concentration
of the substrates (Frankenberger & Tabatabai, 1991a,c). The sensitivity of
the assay procedure that involves determination of the NH/ released is
such that precise results can be obtained with most soils even if the 2-h
incubation time recommended is reduced to 1 h. No hydrolysis of L-as-
paragine, formamide, or urea is found when autoclaved soils are incubated
with solutions of the substrates at 37°C for 2 h under the conditions of the
assay procedures described. L-Glutamine is hydrolyzed slightly during in-
cubation, and appropriate controls should be included to account for the
NH/ -N produced through chemical hydrolysis. The results obtained by
these methods are not affected when the amount of toluene is increased
from 0.2 to 2.0 mL.
The steps involved in the colorimetric method described for urea anal-
ysis in the second method should be strictly adhered to, but any colorimeter
or spectrophotometer that permits color intensity measurement at 500 to
550 nm can be used for the procedure described. The maximum absorption
of the color produced from the reaction of urea, DAM, and TSC as de-
scribed is 527 nm (Douglas & Bremner, 1970a).
Numerous colorimetric procedures for analysis of urea with DAM
have been developed (Yashphe, 1973), but most of these methods are
ENZYMES 801

actually variations of the method developed by Fearon (1939). The main

disadvantages of most methods are: (i) lack of sensitivity, (ii) lack of lin-
earity at low urea concentrations, (iii) lengthiness of procedure, (iv) low
precision, and (v) instability of some of the reagents or the chromogen
compound. The method described suffers from some of these disadvan-
tages, especially from the instability of the chromogen compound and low
reproducibility of the results, including the standard curve. Thus, attempts
have been made to automate the development of color (Searle & Speir,
1976; Douglas et al., 1978).
An oven adjusted to 120°C can be used instead of the water bath,
which reduces the time required for maximum color development to 30
min. It is important that the flask used for color development be cooled
immediately after it is removed from the water bath, because some loss of
color occurs if the flask is not cooled rapidly as specified (Douglas &
Bremner, 1970a). The color developed in the urea analysis described is
photosensitive, but this is not a problem if the color intensity measure-
ments are performed shortly (1 h or less) after color development. Color
intensity measurement can be postponed for several hours if the solutions
of the chromogen complex are stored in the dark.
Calibration curves prepared from urea standards as described show a
linear relationship between urea concentration and color intensity but dif-
fer slightly from day to day. It is recommended, therefore, that urea stan-
dards be included in each series of analyses.
Nitrite interferes with the colorimetric method used for determination
of urea if the concentration of N0 2- -N in the extract analyzed is more than
five times the concentration of urea-No Such concentrations of N0 2- -N are
not expected in soils or in urea-treated soils, but NOi interference can be
eliminated by treating the aliquot of extract taken for urea analysis with 1
mL of 2% (wt/vol) sulfamic acid solution and by allowing the treated
aliquot to stand for 5 min before the addition of the color reagent. The
color reagent is unstable and should be prepared immediately before use.
The other reagents used are stable for several weeks if stored in a refrig-
erator.

37-3.2 Phosphatases

37-3.2.1 Introduction
The general names phosphatases has been used to describe a broad
group of enzymes that catalyze the hydrolysis of both esters and anhydrides
of H 3P0 4 (Schmidt & Laskowski, 1961). The commission on enzymes of
the International Union of Biochemistry has classified all these enzymes
into five major groups (Plorkin & Stotz, 1964). These include the phos-
phoric monoester hydrolases (EC 3.1.3), phosphoric diester hydrolases
(EC 3.1.4), triphosphoric monoester hydrolases (EC 3.1.5), enzymes act-
ing on phosphoryl-containing anhydrides (EC 3.6.1), and enzymes acting
on P-N bonds (EC 3.9), such as the phospho amidase (EC 3.9.1.1).
802 TABATABAI

The phosphomonoesterases, acid phosphatase (orthophosphoric mo-

noester phosphohydrolase, EC 3.1.3.2) and alkaline phosphatase (ortho-
phosphoric monoester phosphohydrolase, EC 3.1.3.1) have been studied
extensively. The enzymes are classified acid and alkaline phosphatases
because they show optimum activities in acid and alkaline ranges, respec-
tively. Because of the importance of these enzymes in soil organic P min-
eralization and plant nutrition, considerable literature has accumulated on
phosphomonoesterases in soils (Speir & Ross, 1978). Most of the litera-
ture, however, is related to acid phosphatase. Consequently this enzyme
has been given a prominent place in several soil biochemistry and enzy-
mology reviews (Cosgrove, 1967; Ramirez-Martinez, 1968; Kiss et aI.,
1975; Speir & Ross, 1978). The general equation of the reaction catalyzed
by acid and alkaline phosphatases is

o 0
I II
R-O-P-O- + H 2 0 - ROH + HO-P-O-. [16]
I I
0- 0-

One of the most interesting properties of acid and alkaline phos-

phatases is their specificities. Although most of the information available
on these enzymes is related to acid phosphatase in soils, these enzymes are
know to hydrolyze a variety of phosphomonoesters. The hydrolysis in soils
of ~-glycerophosphate, phenylphosphate, ~-naphthyl phosphate, and p-ni-
trophenyl phosphate has been reported.
Studies by Eivazi and Tabatabai (1977) and Juma and Tabatabai
(1977, 1978) showed that acid phosphatase is predominant in acid soils and
that alkaline phosphatase is predominant in alkaline soils. The inverse
relationship between phosphatase activity and soil pH suggests that either
the rate of synthesis and release of this enzyme by soil microorganisms or
the stability of this enzyme is related to soil pH. Since higher plants are
devoid of alkaline phosphatase activity (Dick et aI., 1983; Juma & Taba-
tabai, 1988a,b,c), the alkaline phosphatase activity in soils seems derived
totally from microorganisms.
Michaelis constants of soil acid phosphatase have been reported in
several studies (Tabatabai & Bremner, 1971; Cervelli et aI., 1973; Brams
& McLaren, 1974; Thornton & McLaren, 1975; Eivazi & Tabatabai, 1977;
Dick & Tabatabai, 1984). The apparent Km values of acid phosphatase in
soils range from 1.3 to 4.5 mM; values of alkaline phosphatase range from
0.4 to 4.9 mM. Adsorption of phosphatases on clay minerals significantly
alters their kinetic parameters (Dick & Tabatabai, 1987). Similar to other
kinetic studies of soil enzymes, the Km values obtained for phosphomo-
noesterases are affected by shaking the soil-substrate mixture during incu-
bation; normally the values are more uniform among soils when shaking
than when static incubation technique is employed (Tabatabai & Bremner,
1971; Eivazi & Tabatabai, 1977).
ENZYMES 803

Studies have shown that all heavy metals and trace elements inhibit
phosphomonoesterases in soils; the degree of inhibition is related to the
soil and type and concentration of the trace elements used (Tyler, 1974,
1976a,b; Juma & Tabatabai, 1977). Kinetic studies indicate that orthophos-
phate is a competitive inhibitor of acid and alkaline phosphatases in soils
(Juma & Tabatabai, 1978).
Several methods have been proposed for estimation of phosphomo-
noesterase activities of soils (Skujins, 1967). The basic difference is in the
substrate used and consequently in the technique employed in measuring
the product of hydrolysis of the substrate by phosphatase enzymes (Dick &
Tabatabai, 1978b).
Kroll and Kramer (1955) estimated soil phosphatase activity by deter-
mining the phenol released by incubation of soil with phenyl phosphate.
This substrate has been used in several investigations of soil phosphatase
activity (e.g., Kramer, 1957; Kramer & Yerdei, 1959; Halstead, 1964).
Skujins et al. (1962) assayed soil phosphatase activity by a procedure in
which the amount of ~-glycerophosphate hydrolyzed by incubation of soil
with this organic phosphate was estimated by analyses for extractable total
and inorganic P after incubation. This method is tedious and time consum-
ing and has low precision. Ramirez-Martinez and McLaren (1966a) pro-
posed a method involving fluorimetric assay of the ~-naphthol released by
incubation of soil with ~-naphthyl phosphate, but this method is compli-
cated by sorption of ~-naphthol by soil constituents and requires that the
capacity of each soil analyzed to sorb ~-naphthol be determined and al-
lowed for in calculation of the results.
Of the various methods available for assay of phosphatase activity in
soils, the method developed by Tabatabai and Bremner (1969) is the most
rapid, accurate, and precise and is described here. It involves colorimetric
estimation of the p-nitrophenol released when soil is incubated with buff-
ered sodiump-nitrophenyl phosphate solution and toluene. The procedure
used to extract the p-nitrophenol released by phosphatase activity develops
the stable color used to estimate this phenol and gives quantitative recovery
of p-nitrophenol added to soils.
Among the phosphatases in soils, phosphodiesterase (orthophospho-
ricdiester phosphohydrolase, EC 3.1.4.1) is the least studied. Phosphodi-
esterase catalyzes the overall reaction of the type

[17]

where R 1 and R2 represent either alcohol or phenol groups or nucleosides

(Privat de Garilhe, 1967).
The activity of phosphodiesterase has been detected in various plants,
animals, and microorganisms (Browman & Tabatabai, 1978). This enzyme
804 TABATABAI

is perhaps best known for its ability to degrade nucleic acid (Razzel &
Khorana, 1959), and given the demonstrated ability of soils to decompose
added nucleic acids (Pearson et aI., 1941; Rogers, 1942) and the natural
occurrence of these compounds in soils (Anderson, 1967), the potential
role of phosphodiesterase activity in the soil P cycle is obvious.
Studies on inhibition of soil phosphodiesterase have shown that at 5
mM, orthophosphate, EDTA , and citrate are inhibitors of this enzyme.
Similar to inhibition of phosphomonoesterase activity, orthophosphate is a
competitive inhibitor of phosphodiesterase in soils. Kinetics studies indi-
cate that the apparent Km values of this enzyme in soils range from 1.3 to
2.0 mM, the 010 is 1.7, and the average activation energy is 37 kJ/mol
(Browman & Tabatabai, 1978).
_Many factors affect phosphomonoesterase and phosphodiesterase ac-
tivities in soils. For discussion of these factors and information about the
stability and distribution of these enzymes in soils, see Eivazi and Tabata-
bai (1977), Browman and Tabatabai (1978), Juma and Tabatabai (1978),
and Speir and Ross (1978).
Inorganic pyrophosphatase (pyrophosphate phosphohydrolase, Ee
3.6.1.1) catalyzes the hydrolysis of pyrophosphate to orthophosphate. The
overall reaction is

o 0
II II
-O-P-O-P-O- + H 20 --+2HPOa-. [18]
I I
0- 0-

Similar to the other phosphatases described, inorganic 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 by several workers (Gilliam &
Sample, 1968; Hashimoto et aI., 1969; Hossner & Phillips, 1971).
The activity of this enzyme in soils has received special attention,
because its substrate, pyrophosphate, is used as a fertilizer. Although py-
rophosphatase activity in soils has been under investigation for many years,
it was not until recently that an accurate method has become available for
its assay. The information available indicates that pyrophosphatase activity
in soils is optimum at a buffer pH of 8.0. Formaldehyde, fluoride, oxalate,
and carbonate inhibit the activity of this enzyme in soils (Dick & Tabata-
bai, 1978a). Studies by Stott et aI. (1985) showed that pyrophosphatase
activity in soils is inhibited by many metals and that AsOl-, BO~-,
MoOi-, POl-, V02+, and WOi- are competitive inhibitors of this en-
zyme in soils. At low concentrations, however, many metals are activators
of soil pyrophosphatase (Dick & Tabatabai, 1983). Kinetic studies with
surface soils have shown that the apparent Km values of pyrophosphatase
in soils range from 20 to 51 mM, and the activation energy values range
from 32 to 43 kJ/mol (Dick & Tabatabai, 1978a).
ENZYMES 805

Studies by Tabatabai and Dick (1979) showed that similar to other

enzyme activities in soils, pyrophosphatase activity is concentrated in sur-
face soils and decreases with depth; it is significantly correlated with or-
ganic C in surface soils and soil profiles. Pyrophosphatase activity was also
correlated with the percentage clay and mole fraction of Mg/(Mg + Ca) in
water extracts of surface soils and significantly but negatively correlated
with the percentage of CaC03 equivalent in surface soils. Unlike some
other soil enzymes, air-drying of field-moist soils decreases pyrophos-
phatase activity.
Trimetaphosphate (TMP), a cyclic polyphosphate that is not sorbed by
soils, is hydrolyzed by a series of biochemical reactions to yield triphos-
phate, pyrophosphate, and orthophosphate; phosphates that are sorbed by
soils (Busman & Tabatabai, 1984):

[19]

[20]

[21]

The hydrolysis of TMP (reaction [19]) may be catalyzed biochemically by

trimetaphosphatase (trimetaphosphate hydrolase, EC 3.6.1.2) or chemi-
cally by acidic or basic solutions, especially in the presence of certain
cations (Berg & Gordon, 1960; Van Wazer, 1958). Reactions [20] and [21]
are catalyzed by triphosphatase and pyrophosphatase, respectively.
Among the phosphatases involved in these reactions, pyrophosphatase is
the most thoroughly studied (Dick & Tabatabai, 1978a).
Trimetaphosphatase activity has been reported in yeast (Kornberg,
1956; Mattenheimer, 1956; Meyerhof et aI., 1953), intestinal mucosa of
mammals (Berg & Gordon, 1960; Ivey & Shaver, 1977), and in plants
(Stossel et aI., 1981; Pierpoint, 1957). It has been suggested that trimeta-
phosphatase activity occurs in all living organisms (Berg & Gordon, 1960).
Results reported by Blanchar and Hossner (1969) and by Rotini and Car-
loni (1953) on hydrolysis of TMP suggest the presence of trimetaphos-
phatase activity in soils.
The yield of orthophosphate (OP) was used to indicate trimetaphos-
phatase activity in most studies because of the difficulty in determining the
primary product of TMP hydrolysis, triphosphate (TP), in the presence of
other phosphates. However, the dependence of the formation of OP from
TMP on other enzymes (triphosphatase and pyrophosphatase) complicates
the interpretation of the results. In addition, determination of the OP
released is subject to interference by the presence of the other phosphate
compounds (Dick & Tabatabai, 1977b). Until recently, the lack of an
adequate method for determining TMP or TP in soils has limited the
number of studies on trimetaphosphatase activity in soils.
The method described for assay of trimetaphosphatase activity in soils
is based on precipitation of other phosphates in a soil extract with BaCI2 ,
806 TABATABAI

and determining TMP as total P remaining in the solution (Busman &

Tabatabai, 1984, 1985a,b). In addition to its hydrolysis to triphosphate in
soils by the enzyme trimetaphosphatase, TMP is hydrolyzed chemically by
metal ions such as Ca2+ and Mg2-. Therefore, a method is described for
assaying the activity of trimetaphosphatase and for determining the rate of
nonenzymatic (chemical) hydrolysis of TMP in soils. The method involves
incubating 1 g of soil with 3 mL buffered (100 mM of Tris) 25 mM of TMP
at 37°C for 5 h followed by determination of the unaltered substrate.
Steam-sterilized soil is used as a control to account for the amount of TMP
hydrolyzed nonenzymatically.
Trimetaphosphatase activity in soils was maximum at a buffer pH of 8
and a temperature of 40°C. Nonenzymatic hydrolysis ofTMP increased up
to the highest incubation temperature (80°C) tested. Trimetaphosphatase
in three Iowa soils showed apparent Km values ranging from 6.8 to 7.2 mM
of TMP, V max values from 590 to 1200 mg of TMP-hydrolyzed kg- 1 soil 5
h -1, and energy of activation values from 17 to 26 kJ mol- 1 . The activation
energy values of the nonenzymatic hydrolysis ranged from 29 to 39 kJ
mol- 1 • Enzymatic and nonenzymatic hydrolysis rates in the six soils used
ranged from 231 to 928 and from 101 to 888 mg of TMP-P kg- 1 soil 5 h- 1 ,
respectively. Formaldehyde and EDTA inhibited, and toluene had no ef-
fect on trimetaphosphatase activity in soils. The addition of Ca2+ and
Mg2+ increased the nonenzymatic hydrolysis-rates in two soils tested with
Ca2+ being twice as effective as Mg2+ (Busman & Tabatabai, 1985a).
The hydrolysis of polyphosphates in soils and by plant roots has been
reported (Dick & Tabatabai, 1986a,b, 1987; Juma & Tabatabai, 1988a,b).
The methods employed in these studies will not be described here, and the
reader may refer to the references mentioned above.

37-3.2.2 Principles
The procedures described for assay of phosphomonoesterase activities
are based on colorimetric estimation of the p-nitrophenol released by phos-
phatase activity when soil is incubated with buffered (pH 6.5 for acid
phosphatase activity and pH 11 for alkaline phosphate activity) sodium
p-nitrophenyl phosphate solution and toluene. The colorimetric procedure
used for estimation of p-nitrophenol is based on the fact that alkaline
solutions of this phenol have a yellow color (acid solutions of p-nitrophenol
and acid and alkaline solutions of p-nitrophenyl phosphate are colorless).
The CaCI2-NaOH treatment described for extraction of p-nitrophenol after
incubation for assay of acid and alkaline phosphatases serves (i) to stop
phosphatase activity, (ii) to develop the yellow color used to estimate this
phenol, and (iii) to give quantitative recovery of p-nitrophenol from soils.
The principle of assay for phosphodiesterase activity in soils is similar
to that of assay of acid and alkaline phosphatases; the p-nitrophenol re-
leased is extracted and determined colorimetrically. The two procedures
differ, however, in that 0.1 M TRAM buffer pH 12 is used for extraction
ENZYMES 807

of the p-nitrophenol released in the assay of phosphodiesterase activity

instead of the O.S M NaOH used in the assay of phosphomonoesterases.
The reason for this difference is that the substrate of phosphodiesterase,
bis-p-nitrophenyl phosphate (BPNP), is not stable in NaOH solutions;
BPNP is hydrolyzed with time in the presence of NaOH. The CaCI2-
THAM treatment described for extraction of the p-nitrophenol released in
the assay of phosphodiesterase serves the same purpose as that of CaCi2-
NaOH used for extraction of this product in the assay of phosphomono-
esterases.
The assay of inorganic pyrophosphatase activity is based on determi-
nation of the orthophosphate released when soil is incubated with buffered
(pH 8.0) pyrophosphate solution. Generally, there are three problems
associated with the measurement of orthophosphate released by enzymatic
hydrolysis of pyrophosphate during the pyrophosphatase assay: (i) the
orthophosphate released may be sorbed by the soil constituents and there-
fore not extracted; (ii) orthophosphate may continue to be hydrolyzed
from the substrate (pyrophosphate) after extraction from the soil for rea-
sons other than the enzyme (e.g., low pH); and (iii) the presence of py-
rophosphate may interfere with the measurement of orthophosphate. All
these problems must be overcome in any method used to assay the pyro-
phosphatase activity in soils. In the method described, O.S M H 2S0 4 is used
to extract orthophosphate. This reagent gives quantitative recovery of or-
thophosphate added to soils. The colorimetric method used for determi-
nation of the orthophosphate extracted in the presence of pyrophosphate
is specific for orthophosphate. This method involves a rapid formation of
heteropoly blue by the reaction of orthophosphate with molybdate ions in
the presence of ascorbic acid-trichloroacetic acid reagent and complexation
of the excess molybdate ions by a citrate-arsenite reagent to prevent further
formation of blue color from the orthophosphate derived from hydrolysis
of the substrate, pyrophosphate.

37-3.2.3 Assay Methods

37-3.2.3.1 Phosphomonoesterases (Acid and Alkaline Phosphatases)

(Tabatabai & Bremner, 1969; Eivazi & Tabatabai, 1977)

37-3.2.3.1.1 Special Apparatus

1. Incubation flasks, SO-mL Erlenmeyer flasks fitted with no. 2 stop-
pers.
2. Incubator, ordinary incubator, or temperature-controlled water
bath.
3. Colorimeter or spectrophotometer, Klett-Summerson photoelec-
tric colorimeter fitted with a blue (no. 42) filter or a spectropho-
tometer than can be adjusted to a wavelength of 400 to 420 om.
808 TABATABAI

37-3.2.3.1.2 Reagents
1. Toluene, Fisher certified reagent (Fisher Scientific Co., Chicago).
2. Modified universal buffer (MUB) stock solution: Dissolve 12.1 g of
tris(hydroxymethyl)aminomethane (THAM), 11.6 g of maleic acid,
14.0 g of citric acid, and 6.3 g of boric acid (H3B03) in 488 mL of
1 N sodium hydroxide (NaOH) and dilute the solution to 1 L with
water. Store it in a refrigerator.
3. Modified universal buffer, pH 6.5 and 11: Place 200 mL of MUB
stock solution in a 500-mL beaker containing a magnetic stirring
bar, and place the beaker on a magnetic stirrer. Titrate the solution
to pH 6.5 with 0.1 M hydrochloric acid (HCI), and adjust the vol-
ume to 1 L with water. Titrate another 200 mL of the MUB stock
solution to pH 11 by using 0.1 M NaOH, and adjust the volume to
1 L with water.
4. p-Nitrophenyl phosphate solution, 0.05 M: Dissolve 0.840 g of
disodium p-nitrophenyl phosphate tetrahydrate (Sigma 104, Sigma
Chemical Co., St. Louis, MO) in about 40 mL of MUB pH 6.5
(for assay of acid phosphatase) or pH 11 (for assay of alkaline
phosphatase), and dilute the solution to 50 with MUB of the same
pH. Store the solution in a refrigerator.
5. Calcium chloride (CaCI 2) 0.5 M: Dissolve 73.5 g of CaCI2 ·2H20 in
about 700 mL of water, and dilute the volume to 1 L with water.
6. Sodium hydroxide (NaOH), 0.5 M: Dissolve 20 g of NaOH in
about 700 mL of water, and dilute the volume to 1 L with water.
7. Standard p-nitrophenol solution: Dissolve 1.0 g of p-nitrophenol in
about 700 mL of water, and dilute the solution to 1 L with water.
Store the solution in a refrigerator.

37-3.2.3.1.3 Procedure
Place 1 g of soil « 2 mm) in a 50-mL Erlenmeyer flask, add 0.2 mL
of toluene, 4 mL of MUB (pH 6.5 for assay of acid phosphatase or pH 11
for assay of alkaline phosphatase), 1 mL of p-nitrophenyl phosphate solu-
tion made in the same buffer, and swirl the flask for a few seconds to mix
the contents. Stopper the flask, and place it in an incubator at 37°C. After
1 h, remove the stopper, add 1 mL of 0.5 M CaCl2 and 4 mL of 0.5 M
NaOH, swirl the flask for a few seconds, and filter the soil suspension
through a Whatman no. 2v folded filter paper. Measure the yellow color
intensity of the filtrate with a Klett-Summerson photoelectric colorimeter.
Calculate the p-nitrophenol content of the filtrate by reference to a cali-
bration graph plotted from the results obtained with standards containing
0, 10, 20, 30, 40, and 50 JA.g of p-nitrophenol. To prepare this graph, dilute
1 mL of the standard p-nitrophenol solution to 100 mL in a volumetric flask
and mix the solution thoroughly. Then pipette 0-, 1-,2-,3-,4-, and 5-mL
aliquots of this diluted standard solution into a 50-mL Erlenmeyer flasks,
adjust the volume to 5 mL by addition of water, and proceed as described
ENZYMES 809

for p-nitrophenol analysis of the incubated soil sample (i.e., add 1 mL of

0.5 M CaCl2 and 4 mL of 0.5 M of NaOH, mix and filter the resultant
suspension). If the color intensity of the filtrate exceeds that of 50 Ilg of the
p-nitrophenol standard, an aliquot of the filtrate should be diluted with
water until the colorimeter reading falls within the limits of the calibration
graph.
Controls should be performed with each soil analyzed to allow for
color not derived from p-nitrophenol released by phosphatase activity. To
perform controls, follow the procedure described for assay of phosphatase
activity, but make the addition of 1 mL of PNP solution after the additions
of 0.5 M CaCl2 and 4 mL of 0.5 M NaOH (i.e., immediately before filtra-
tion of the soil suspension).

37-3.2.3.2 Phosphodiesterase (Browman & Tabatabai, 1978)

37-3.2.3.2.1 Special Apparatus

1. Erlenmeyer flasks (50 mL), stoppers (no. 2), incubator, and Klett-
Summerson photoelectric colorimeter described in 37-3.2.3.1.

37-3.2.3.2.2 Reagents
1. Toluene, Fisher certified reagent (Fisher Scientific Co., Chicago).
2. Tris(hydroxymethyl)aminomethane (TRAM) buffer, 0.05 M, pH
8.0: Dissolve 6.1 g of THAM (Fisher certified reagent, Fisher Sci-
entific Co., Chicago) in about 800 mLofwater, adjust the pH to 8.0
by titration with approximately 0.1 M H 2S04 , and dilute the solu-
tion to 1 L with water.
3. Bis-p-nitrophenyl phosphate (BPNP) solution, 0.05 M: Dissolve
0.906 g of sodium, bis-p-nitrophenyl phosphate in about 40 mL of
THAM buffer pH 8.0, and dilute the volume to 50 mL with buffer.
Store the solution in a refrigerator.
4. Calcium chloride (CaCI2 solution, 0.5 M: Prepare as described for
reagent 5 in 37-3.2.3.1.2.
5. Tris(hydroxymethyl)aminomethane-sodium hydroxide (THAM-
NaOH) extractant solution, 0.1 M THAM (Fisher certified re-
agent, Fisher Scientific Co., Chicago), pH 12: Dissolve 12.2 g of
TRAM in about 800 mL of water, adjust the pH of the solution to
12 by titration with 0.5 M NaOH. and dilute the volume to 1 L with
water.
6. Tris(hydroxymethyl)aminomethane (TRAM) diluent, 0.1 M, pH
about 10: Dissolve 12.2 g of TRAM (Fisher certified reagent,
Fisher Scientific Co., Chicago) in about 800 mL of water, and
adjust the volume to 1 L with water.
7. Standard p-nitrophenol solution: Prepare as described for reagent
7 in 37-3.2.3.1.2.
810 TABATABAI

37-3.2.3.2.3 Procedure
Place 1 g of soil ( < 2 mm) in a 50-mL Erlenmeyer flask, and add 0.2
mL of toluene, 4 mL of THAM buffer pH 8.0, and 1 mL of BPNP solution.
Swirl the flask for a few seconds to mix the contents. Stopper the flask, and
incubate it at 37°C. After 1 h, remove the stopper, add 1 mL of 0.5 M
CaCl2 and 4 mL of THAM-NaOH extractant solution, swirl the flask for a
few seconds, and filter the suspension through a Whatman No. 2v folded
filter paper. Measure the yellow color intensity of the filtrate with a Klett-
Summerson photoelectric colorimeter. Calculate the p-nitrophenol content
of the filtrate by reference to a calibration graph prepared as described in
section 37-3.2.3.1.3.
If the color intensity of the filtrate exceeds that of 50 ~g of p-nitro-
phenol standard, an aliquot of the filtrate should be diluted with 0.1 M
THAM pH about 10 until the colorimeter reading falls within the limits of
the calibration graph.
Controls should be performed with each soil analyzed to allow for
color not derived from PN released by phosphodiesterase activity. To per-
form controls, follow the procedure described for assay of phosphodi-
esterase activity, but make the addition of 1 mL of BPNP solution after the
addition of 0.5 M CaCl2 and 4 mL of 0.1 M THAM-NaOH (i.e., imme-
diately before filtration of the soil suspension).
37-3.2.3.3 Inorganic Pyrophosphatase (Dick & Tabatabai, 1977b,
1978a)

37-3.2.3.3.1 Special Apparatus

1. Centrifuge tubes, 50 mL, plastic.
2. Incubator, ordinary incubator, or temperature-controlled water
bath.
3. Shaker, end-to-end shaker.
4. Centrifuge, high speed, 12000 rpm (17390 g).
5. Spectrophotometer or colorimeter.

37-3.2.3.3.2 Reagents
1. Sodium hydroxide (NaOH), 1 M.
2. Modified universal buffer stock solution: Prepared as described for
reagent 2 in section 37-3.2.3.1.2.
3. Pyrophosphate solution, 50 mM: Dissolve 2.23 g of sodium pyro-
phosphate decahydrate (Na4P2 0 7 ·10H2 0) (Matheson Coleman
and Bell Manufacturing Chemists, Norwood, OH) in 20 mL of
MUB stock solution, titrate the solution to pH 8 with 0.1 M HCI,
and dilute the volume to 100 mL with water. This reagent should be
prepared daily.
4. Modified universal buffer working solution, pH 8.0: Titrate 200 mL
of the MUB stock solution to pH 8.0 with 0.1 M HCl, and dilute the
volume to 1 L with water.
ENZYMES 811

5. Sulfuric acid (H2S04), 0.5 M: Add 250 mL of concentrated H 2S04

to 8 L of water, and dilute the volume to 9 L with water.
6. Ascorbic acid (0.1 M)-trichloroacetic acid (0.5 M) reagent (reagent
A): Dissolve 8.8 g of ascorbic acid and 41 g of trichloroacetic acid
(Fisher certified reagent) in about 400 mL of water, and dilute the
volume to 500 mL with water. This reagent should be prepared
daily.
7. Ammonium molybdate tetrahydrate [(NH4)6M07024 4H20] (0.015
M) reagent (reagent B): Dissolve 9.3 g of ammonium molybdate
(J.T. Backer Chemical Co., Phillipsburg, NJ) in about 450 mL of
water, and adjust the volume to 500 mL with water.
8. Sodium citrate (0.15 M)-sodium arsenite (0.3 M)-acetic acid
(7.5%) reagent (reagent C): Dissolve 44.1 g of sodium citrate and
39 g of sodium arsenite in about 800 mL of water, add 75 mL of
glacial acetic acid (99.9%), and adjust the volume to 1 L with
water.
9. Standard phosphate stock solution: Dissolve 0.4390 g of potassium
dihydrogen phosphate (KH2P04) in about 700 mL of water, and
dilute the volume to 1 L with water. This solution contains 100 I-lg
of POg- -P/mL.
37-3.2.3.3.3 Procedure
Place 1 g of soil ( < 2 mm) in a 50-mL plastic centrifuge tube, add 3 mL
of 50 mM pyrophosphate 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 of MUB pH 8 and 25 mL
of 0.5 M H 2S0 4. Stopper the tube, and shake it horizontally in a reciprocal
shaker for 3 min. Centrifuge the soil suspension for 30 s at 17390 g, and
immediately take an aliquot of the supernatant for POg--P analysis.
To analyze for the inorganic phosphate(Pi) released by inorganic py-
rophosphatase, pipette a 1-mL aliquot of the supernatant solution into a
25-mL volumetric flask containing 10 mL of reagent A, immediately add 2
mL of reagent Band 5 mL of reagent C, and adjust the volume with water
(swirl the flask to mix the contents after addition of the sample aliquot and
each of reagents B and C). After 15 min, measure the absorbance of the
heteropoly blue color developed using a spectrophotometer adjusted to a
wavelength of 700 nm.
Calculate the POg--P content of the aliquot analyzed by reference to
a calibration graph plotted from the results obtained with standards con-
taining 0,5, 10, 15,20, and 25 I-lg of pog- -Po To prepare this graph, pipette
0-, 5-, 10-, 15-, 20-, and 25-mL aliquots of the standard pog--P stock
solution into 100-mL volumetric flasks, make up the volumes with water,
and mix thoroughly. Then analyze 1-mL aliquots of these diluted standards
as described for analysis of POg--P in the aliquot of the incubated sample.
Controls should be included with each soil analyzed to allow for
POg- -P not derived from pyrophosphate through pyrophosphatase activ-
ity. To perform controls, add 3 mL of MUB pH 8.0 to 1 g of soil, and
812 TABATABAI

incubate for 5 h. After incubation, add 3 mL of 50 mM of pyrophosphate

solution, immediately add 25 mL of 0.5 M H 2 S04 , and then extract and
analyze for POl- -P as described for the assay sample.
37-3.2.3.4 Trimetaphosphatase (Busman & Tabatabai, 1984, 1985a)

37-3.2.3.4.1 Special Apparatus

1. Centrifuge tubes, 50 mL, plastic.
2. Incubator, ordinary incubator, or temperature-controlled water
bath.
3. Centrifuge, high speed, 12000 rpm (17390 g).
4. Spectrophotometer or colorimeter.

37-3.2.3.4.2 Reagents
1. Trimetaphosphatate (TMP) solution (25 mM): Dissolve 1.53 g of
Na3P309 (practical grade, Sigma Chemical Co., St. Louis, MO)
and 2.42 g of tris(hydroxymethyl)aminomethane (THAM) in about
100 mL of water, adjust the pH to 8.0 with 0.2 M of HCI, and make
up the volume to 200 mL with water.
2. Trimetaphosphate stock solution (100 mM): Dissolve 3.06 g of
Na3P309 (practical grade, Sigma Chemical Co., St. Louis, MO) in
about 80 mL of water, and make up the volume to 100 mL with
water.
3. Trimetaphosphate working solution: Dissolve 1.21 g of THAM in
about 25 mL of water, add 1, 5, 10,25, or 50 mL of the TMP stock
solution (to make 1, 5, 10, 25, or 50 mM of TMP, respectively),
adjust the pH to 8.0 with 0.2 M of HCI, and make up the volume
to 100 mL with water.
4. Barium chloride solution (100 mM): Dissolve 24.4 g of BaCl2 in
about 800 mL of water, and adjust the volume to 1 L with water.
5. Ascorbic acid (0.1 M)-trichloroacetic acid (0.5 M) reagent (reagent
A), ammonium molybdate tetrahydrate reagent (reagent B), so-
dium citrate (0.15 M)-sodium arsenite (0.3 M)-acetic acid (7.5%)
reagent (reagent C), and standard phosphate stock solution: Pre-
pare as described in section 37-3.2.3.3.2.
6. Modified Murphy-Riley reagent: Prepare as described by Dick and
Tabatabai (1977a) but with 4 M of HCI substituted for 2.5 M of
H 2S0 4 ,

37-3.2.3.4.3 Procedure
Place 1 g of soil ( < 2 mm) in a 50-mL plastic centrifuge tube, add 3 mL
of 25 mM of TMP solution, swirl the tube for a few seconds to mix the
contents. Stopper the tube, and incubate at 37°C. After 5 h, remove the
stopper, mix on a vortex stirrer and during mixing add 10 mL of 100 mM
of BaCl2 slowly to precipitate other phosphate compounds. Centrifuge the
tube at 12 000 x g for 5 min, and immediately remove aliquots of the
ENZYMES 813

supernatant for determination of orthophosphate and TMP by placing an

aliquot containing < 25 Ilg of orthophosphate-P into a 25-mL volumetric
flask and another aliquot (1-5 mL) containing < 1 mg P into a 50-mL
volumetric flask, respectively. Determine orthophosphate-P as described
in section 37-3.2.3.3.3.
Acidify the aliquot removed for determination of TMP with sufficient
2 M of HCl to bring the concentration to 1 M with respect to HCI and heat
the flask on a steam plate (85°C) for 1 h to hydrolyze the TMP to ortho-
phosphate. After cooling to room temperature, adjust the volume to 50 mL
with water. Determine the orthophosphate content of this flask by analyz-
ing an aliquot by the Murphy-Riley reagent after it was neutralized with 1
M of NaOH as described by Dick and Tabatabai (1977a).
Two types of controls should be performed. In one control, the above
procedure should be followed with steam-sterilized soil. A second control
should be performed with each set of assays to determine precisely the
amount of TMP added to the soils. To perform this control, follow the
above procedures, but without soil.
The amount of TMP-P hydrolyzed enzymatically is calculated by sub-
tracting the amount of TMP-P remaining in the assay tube (i.e., the tube
containing non sterilized soil) from the amount remaining in the control
tube (i.e., the tube containing steam-sterilized soil). The amount ofTMP-P
hydrolyzed nonenzymatically is calculated by subtracting the amount of
TMP-P remaining in the control tube (i.e., the tube containing sterilized
soil) from the amount of P in the control tube containing no soil.

37-3.2.4 Comments
The solutions of the substrates used for assay of phosphomono-
esterases and phosphodiesterase are stable for several days if stored in a
refrigerator. The compounds used for assay of these enzymes are artificial
substrates; are not expected to be found in soils. The dry substrates should
be stored in a desiccator in a freezer. The standard p-nitrophenol solution
is stable for a few weeks if stored in a refrigerator.
It is necessary to add CaCl 2 to prevent dispersion of clay and extrac-
tion of soil organic matter during the treatment with NaOH in assay of
phosphomonoesterases (dispersion of clay complicates filtration, and the
dark-colored organic matter extracted with NaOH interferes with colori-
metric analysis for p-nitrophenol). The procedures described give quanti-
tative recovery of p-nitrophenol added to soils. In assays of acid and
alkaline phosphatases, the control analysis is so designed that it allows for
the presence of trace amounts of p-nitrophenol in some commercial sam-
ples of p-nitrophenyl phosphate and for extraction of trace amounts of
colored soil material by the CaCI2-NaOH treatment used for extraction of
p-nitrophenol. No chemical hydrolysis of p-nitrophenyl phosphate is de-
tected under the conditions of the assay procedure described.
Acid and alkaline phosphatase and phosphodiesterase activity values
are not affected by the amount of toluene added (0.1-1.0 mL). Toluene,
814 TABATABAI

however, increases slightly the observed activities. Air-drying of field-

moist soils results in increases in acid phosphatase activity and a decrease
in alkaline phosphatase activity (Eivazi & Tabatabai, 1977).
The substrate concentrations in the incubation mixtures used for assay
of phosphomonoesterases and phosphodiesterase are about 5 to 10 times
greater than the Km values of these enzymes in soils. If desired, these
substrate concentrations can be changed to meet the objectives of the
assay.
The assay for phosphodiesterase should not be carried out for a long
time because the risk from hydrolysis of the second product (p-nitrophenyl
phosphate) produced from the action of phosphodiesterase on BPNP in-
creases as incubation time increases. This is especially true in the absence
of buffer (Browman & Tabatabai, 1978). This risk is minimal, however,
under the conditions of the procedure described.
When assay for pyrophosphatase activity, it is important that the ex-
traction and analysis of the orthophosphate released from pyrophospha-
tase be carried out immediately, because pyrophosphate hydrolyzes slowly
with time in the presence of the extractant (0.5 M H 2S04), Also, the steps
involved in determination of the orthophosphate in the presence of pyro-
phosphate should be adhered to. The reagents described should be tested
for orthophosphate, because the presence of orthophosphate in any of the
reagents may give a high value for the controls.
For the assay of trimetaphosphatase, a concentration of 25 mM of
TMP was chosen because at least 20 mM is necessary for enzyme saturation
and much higher concentrations would be detrimental to the precision of
the assay (i.e., with little TMP hydrolyzed a high amount would remain to
be determined). Tests performed on several soils showed that TMP can be
recovered quantitatively by the method described and that the enzymatic
and nonenzymatic hydrolysis of TMP are proportional to time of incuba-
tion (up to 5 h) and to the amount of soil used (up to 1.5 g of soil) (Busman
& Tabatabai, 1984, 1985a). Other studies showed that treatment of soils
with formaldehyde, EDTA, mercuric chloride, sodium nitrate, ascorbic
acid, and orthophosphate inhibited trimetaphosphatase activity in soils.
The activity of this enzyme is noncompetitively inhibited by EDTA, PO]-,
and analogues of POl-, (MoO~-, WO~-, V02+, AsOl-, and B40~-).
Treatment of soils with formaldehyde, EDTA, ascorbic acid, orthophos-
phate, pyrophosphate, or triphosphate decrease the nonenzymatic hydrol-
ysis of TMP, whereas treatment of soils with K2S04, NH4Cl, CaCI2 , and
MgCl2 increases the rate of nonenzymatic hydrolysis (Busman & Tabata-
bai, 1985b).
37-3.3 Arylsulfatase
37-3.3.1 Introduction
Several types of sulfatases occur in nature. They have been classified
according to the type of organic sulfate esters they hydrolyze, with the
following main groups recognized: arylsulfatases, alkylsulfatases, steroid
ENZYMES 815

sulfatases, glucosulfatases, chondrosulfatases, and myrosulfatases (From-

ageot, 1950; Roy, 1960). Arylsulfatase (arylsulfate sulfohydrolase, EC
3.1.6.1) is the enzyme that catalyzes the hydrolysis of an arylsulfate anion
by fission of the O-S bond (Spencer, 1958). The reaction

[22]

is irreversible, and there is no evidence that any SO,i- acceptor other than
water can be used or that any metal ion is required for its catalytic function.
This enzyme was first discovered in 1911 in purple snails by Derrien2 , and
it has been detected in plants, animals, and microorganisms (Nicholls &
Roy, 1971). Because this enzyme was the first sulfatase to be detected in
nature, it has received more attention than other groups of sulfatases.
Arylsulfatase was first detected in Iowa soils by Tabatabai and Brem-
ner (1970a). Since then it has been detected in other soils around the world
and in marine sediments (Cooper, 1972; Chandramohan et aI., 1974; Speir
& Ross, 1975; Kowalenko & Lowe, 1975; Thornton & McLaren, 1975;
Houghton & Rose, 1976). Arylsulfatase is believed to be partly responsible
for S cycling in soils. The suggested role of this enzyme in S mineralization
is largely derived from studies showing that between 40 and 70% (avg.
50%) of the total S in surface soils of temperate regions is reduced to H 2S
by HI and is converted to inorganic So,i- with hot alkali (Freney, 1961;
Tabatabai & Bremner, 1972b; Neptune et aI., 1975). These findings sug-
gested that this fraction of S in surface soils is present in the form of ester
sulfate (organic sulfates) and suggested further that arylsulfatase may play
an important role in the processes whereby organic soil S is mineralized and
made available for plant growth.
The procedure developed by Tabatabai and Bremner (1970a), and
which has been used in all other studies in assay of arylsulfatase activity in
soils, involves determination of the p-nitrophenol released when the soil
sample is incubated with buffered p-nitrophenyl sulfate solution and tolu-
ene at 37 °C for 1 h. Tests have indicated that arylsulfatase activity in soils
is inactivated at temperatures ranging from 60 to 70 0c. The Km values of
this soil enzyme range from 0.2 to 5.7 mM. Its determination is affected by
shaking the soil-buffer-substrate mixture during incubation; usually the Km
values are lower and more uniform among soils when the shaking incuba-
tion rather than the static technique is used (Tabatabai & Bremner, 1971;
Thornton & McLaren, 1975).
Studies of factors affecting arylsulfatase activity have shown that the
activity of this enzyme decreases with depth in soil profiles, which is asso-
ciated with a decrease in organic matter content. Also, arylsulfatase activ-
ity is significantly correlated with the organic matter content of surface soils
differing markedly in chemical and physical properties (Tabatabai & Brem-
ner, 1970b). Recent studies indicate that several trace elements inhibit
arylsulfatase activity in soils and that the inhibition by MoO,i-, WO,i-,

2Biochemical information. Vol. 1. 1973. Boehringer Mannheim GmbH. p. 48.

816 TABATABAI

As01- , and P01- shows competitive kinetics. At 25 !Ullol/g of soil, com-

mon anions of soils such as N0 2- N0 3-, Cl-, and SO~- are not inhibitors
of this enzyme (Al-Khafaji & Tabatabai, 1979).
Generally, air-drying of field-moist soils results in an increase in aryl-
sulfatase activity. A possible explanation of this increase is that rewetting
of air-dry soil causes a breakdown of aggregates, thus increasing the ac-
cessibility of arylsulfatase to the substrate (Tabatabai & Bremner, 1970b).
Other factors that may affect arylsulfatase activity in soils are summarized
by Speir & Ross (1978).

37-3.3.2 Principles
The principles of the method described are similar to those of the assay
for phosphomonoesterases (acid and alkaline phosphatases) described in
section 37-3.2.2. The method is based on colorimetric determination of
p-nitrophenol released by arylsulfatase activity when soil is incubated with
buffered (pH 5.8) potassium p-nitrophenyl sulfate solution and toluene.
Usually the soil-buffer-substrate mixture is incubated at 37°C for 1 h. The
p-nitrophenol released is extracted by filtration after the addition of CaCl2
and NaOH reagents. The colorimetric procedure used for determination of
p-nitrophenol depends on the fact that alkaline solutions of this phenol
have a yellow color (acid solutions of p-nitrophenol and acid and alkaline
solutions of p-nitrophenyl sulfate are colorless). The CaCI2-NaOH treat-
ment is employed for the same purpose as that described in section 37-
3.2.2.

37-3.3.3 Assay Method (Tabatabai & Bremner, 1970a)

37-3.3.3.1 Special Apparatus
1. Erlenmeyer flasks (50 mL), stoppers (no. 2), incubator, and Klett-
Summerson photoelectric colorimeter described in section 37-
3.2.3.1.1.
37-3.3.3.2 Reagents
1. Toluene, Fisher certified reagent (Fisher Scientific Co., Chicago).
2. Acetate buffer, 0.5 M, pH 5.8: Dissolve 68 g of sodium acetate
trihydrate in about 700 mL of water, add 1.70 mL of glacial acetic
acid (99%), and dilute the volume to 1 L with water.
3. p-Nitrophenyl sulfate solution, 0.05 M: Dissolve 0.614 g of potas-
siump-nitrophenyl sulfate (Sigma Chemical Co., St. Louis, MO) in
about 40 mL of acetate buffer, and dilute the solution to 50 mL with
buffer. Store this solution in a refrigerator.
4. Calcium chloride (CaCI2 , 0.5 M), sodium hydroxyide (NaOH, 0.5
M), and standard p-nitrophenol solution: Prepare as described in
section 37-3.2.3.1.2.
37-3.3.3.3 Procedure. Place 1 g of soil ( < 2 mm) in a 50-mL Erlen-
meyer flask, add 0.25 mL of toluene, 4 mL of acetate buffer, and 1 mL of
ENZYMES 817

p-nitrophenyl sulfate solution, and swirl the flask for a few seconds to mix
the contents. Stopper the flask, and place it in an incubator at 37°C. After
1 h, remove the stopper, add 1 mL of 0.5 M CaCl2 and 4 mL of 0.5 M
NaOH, swirl the flask for a few seconds, and filter the soil suspension
through a Whatman No. 2v folded filter paper. Measure the yellow color
intensity of the filtrate with a Klett-Summerson photoelectric colorimeter
fitted with a blue (no. 42) filter. Calculate the p-nitrophenol concentration
by reference to a calibration graph prepared from standard p-nitrophenol
as described in section 37-3.2.3.1.3. if the color intensity of the filtrate
exceeds the limit of the calibration graph, dilute an aliquot of the filtrate
with water until the colorimeter reading falls within the limits of the graph.
Controls should be performed with each soil analyzed to allow for
color not derived from p-nitrophenol released by arylsulfatase activity. To
perform controls, follow the procedure described for assay of aryl sulfatase
activity, but make the addition of 1 mL of p-nitrophenyl sulfate solution
after the addition of 1 mL of 0.5 M CaCl2 and 4 mL of 0.5 M NaOH (i.e.,
immediately before filtration of the soil suspension).

37-3.3.4 Comments
Although several compounds (e.g., potassium phenyl sulfate, potas-
sium nitrocatechol sulfate, and potassium phenolphthalein sulfate) can
serve as substrates for arylsulfatase activity, it is difficult to extract the
phenolic compounds released by enzymatic hydrolysis of these compounds.
The procedure described gives quantitative (99-100%) recovery of p-ni-
trophenol added to soils. It is necessary to add CaCl2 to prevent dispersion
of clay and extraction of soil organic matter during the treatment with
NaOH (section 37-3.2.4).
The control allows for the presence of trace amounts of p-nitrophenol
in some commercial samples of p-nitrophenyl sulfate and for extraction of
trace amounts of colored soil material by the CaCI2-NaOH treatment. No
chemical hydrolysis of p-nitrophenyl sulfate is detected under the condi-
tions of the assay procedure described.
Arylsulfatase activity values by the procedure described are lower if
water is used instead of pH 5.8 acetate buffer. The difference between the
values obtained by using water and buffer is related to the difference
between the soil pH and optimum pH (6.2) observed for soil arylsulfatase
activity (Tabatabai & Bremner, 1970a).
The substrate concentration in the incubated mixture (10 mM) rec-
ommended is much higher (5- to lO-fold) than the Km values of aryl sulfa-
tase in most soils. If desired, this substrate concentration can be changed
to meet the objective of the assay.
Choice of buffer and buffer pH in the method described is based on
findings of Tabatabai and Bremner (1970a) showing that the amount of
p-nitrophenol released by incubation of soil with buffered p-nitrophenyl
sulfate solution is considerably higher in acetate buffer than in other buff-
ers they tested and that maximal enzymatic hydrolysis of p-nitrophenyl
818 TABATABAI

sulfate occurs with 0.5 M NaOAc buffer having pH 6.2. However, because
the buffering poise ofthe acetate buffer is greater at pH 5.8 than at 6.2 and
because the amount of p-nitrophenol released at pH 5.8 is not markedly
different from that released at pH 6.2, a buffer pH of 5.8 is recommended.
Arylsulfatase activity values are not affected by the amount of toluene
added (0.1-1.0 mL). Toluene, however, increases the observed activity of
this enzyme in soils.

37-3.4 Rhodanese

37-3.4.1 Introduction
Rhodanese (thiosulfate-cyanide sulfurtransferase, EC 2.8.1.1) is the
enzyme that catalyzes the formation of thiocyanate (SCN-) from thiosul-
fate (S20~-) and cyanide (CN-):

[23]

Lang (1933) discovered rhodanese in animal tissues, and since then, it has
been shown that it is widely distributed in nature. Rhodanese activity was
detected in soils (Tabatabai & Singh, 1976) and its possible importance in
the S cycle was demonstrated by Nor and Tabatabai (1977), who found that
S20~- is an intermediate S compound produced during oxidation of ele-
mental S in soils. The role of rhodanese activity in So oxidation has been
reported (Deng & Dick, 1990; Dick & Deng, 1991). Rhodanese activity is
correlated with organic C in soils, and its activity is affected by various soil
pretreatments and inorganic salts (Singh & Tabatabai, 1978) and water-
logging (Ray et al., 1985). Studies on kinetic parameters of the rhodanese-
catalyzed reaction in soils showed that the Km values 'of S20~- and CN- for
this enzyme are similar to those for the same enzyme isolated from other
biological systems (Tabatabai & Singh, 1979). The Km values of S20~- and
CN- for rhodanese activity in five soils ranged from 1.2 to 10.3 and from
2.5 to 10.2 mM, respectively. The Vmax values ranged from 511 to 1431
nmol of SCN produced g-1 of soil h -1. The activation energy values ranged
from 21.6 to 34 kJ mol-I, and the average 010 for temperatures ranging
from 10 to 60°C ranged from 1.25 to 1.45 (overall avg, 1.37).

37-3.4.2 Principles
The method described is based on colorimetric determination of the
SCN- produced by rhodanese activity when soil is incubated with buffered
(pH 6.0) Na2S203' KCN solutions, and toluene. Incubation is carried out
at 37°C for 1 h, and the reaction is terminated by the addition of a solution
containing CaS04 and formaldehyde. The SCN- produced is extracted by
filtration. The procedure used for determination of the SCN- produced is
based on the reaction of SCN- with Fe3+ in acidic medium to form an
Fe-SCN colored complex, which is measured spectrophotometrically at 460
ENZYMES 819

nm. The color is stable for at least 1 h. The formaldehyde present in the
extractant stops rhodanese activity and prevents formation of the blue
color due to an Fe-S203- complex.
No SCN- formation is expected with Na2S203 and KCN when incu-
bated without soil under the conditions of the assay procedure described.
Copper ions, however, catalyze the reaction of S20j- and CN- with the
formation of SCN- (Nor & Tabatabai, 1975, 1976). Therefore, autoclaved
soil samples should be included in the assay for rhodanese to allow for any
chemical analysis.

37-3.4.3 Assay Method (Tabatabai & Singh, 1976)

37-3.4.3.1 Special Apparatus

1. Incubation flasks, stoppers, incubators, and spectrophotometer as
described in section 37-3.2.3.1.1.

37-3.4.3.2 Reagents
1. Toluene, Fisher certified reagent (Fisher Scientific Co., Chicago).
2. Tris(hydroxymethyl)aminomethane-sulfuric acid (THAM-H2S04 )
buffer (0.05 M, pH 6.0): Dissolve 6.1 g of THAM (Fisher certified
reagent, Fisher Scientific Co., Chicago) in about 600 mL of water,
bring the pH of the solution to 6.0 by titration with approximately
0.2 M H 2S0 4 , and dilute with water to 1 L.
3. Sodium thiosulfate (Na2S203) solution (0.1 M): Dissolve 24.9 g of
Na2S203 and 6.1 g of tris(hydroxymethyl)aminomethane (THAM)
in about 600 mL of water, bring the pH of the solution to 6.0 by
titration with approximately 0.2 N sulfuric acid (H2S0 4 ) and dilute
with water to 1 L.
4. Potassium cyanide (KCN) solution (0.1 M): Dissolve 6.5 g of KCN
and 6.1 g of tris(hydroxymethyl)aminomethane (THAM) in about
600 mL of water, bring the pH of the solution to 6.0 by titration
with approximately 0.2 N sulfuric acid (H 2S0 4), and dilute with
water to 1 L.
5. Formaldehyde solution (37%), Fisher certified reagent.
6. Calcium sulfate-formaldehyde solution: Dissolve 2.2 g of CaS0 4
2H20 in about 900 mL of water, and adjust the volume to 1 L with
water. Mix 100 mL of formaldehyde with 900 mL of the calcium
sulfate solution.
7. Ferric nitrate-nitric acid solution [0.25 M Fe (N0 3h·9H 20-3.1 M
HN0 3]: Dissolve 50 g of ferric nitrate nanohydrate in 100 mL of
concentrated HN03 (analytical reagent grade, sp. grade 1.42), add
this solution to 300 mL of water, and adjust the volume to 500 mL
with water.
Standard thiocyanate stock solution (20 mM): Dissolve 1.621 g of
sodium thiocyanate (NaSCN) in about 800 mL of water, and dilute
the solution to 1 L. One milliliter of this solution contains 20 !J,mol
of NaSCN.
820 TABATABAI

37-3.4.3.3 Procedure. Place 4 g of soil ( < 2 mm) in a 50-mL Erlen-

meyer flask, add 0.5 mL of toluene, 8 mL of THAM buffer, 1 mL of 0.1 M
Na2S203, and 1 mL of 0.1 M KCN, and swirl the flask for a few seconds to
mix the contents. Stopper the flask and place it in an incubator adjusted at
37°C. After 1 h, remove the stopper, and add 10 mL of CaS04-formal-
dehyde solution. Swirl the flask for a few seconds, and filter the soil sus-
pension through a Whatman No. 2v folded filter paper. Pipette 5 mL of the
filtrate into a test tube, add 1 mL of the ferric nitrate reagent, and measure
the absorbance of the reddish brown color of the Fe-SCN complex formed
with a spectrophotometer adjusted to wavelength of 460 nm.
Calculate the thiocyanate content of the filtrate by reference to a
calibration graph plotted from the results obtained with standards contain-
ing 0, 200, 400, 600, 800, and 1000 nmol of thiocyanate. To prepare this
graph, dilute 0, 2, 4, 6, 8, and 10 mL of the standard thiocyanate stock
solution to 1 L in volumetric flasks, and mix the solutions thoroughly. Then
pipette 5-mL aliquots of these diluted standards into test tubes, and pro-
ceed as described for analysis of the thiocyanate in the soil filtrate. If the
color intensity of the filtrate exceeds that of the highest thiocyanate
standard, take a smaller aliquot of the filtrate and adjust the volume to 5
mL with water before adding 1 mL of the ferric nitrate reagent.
Controls should be performed with each soil analyzed to allow for the
slight yellow color derived from soil. To perform controls, follow the pro-
cedure described for assay of rhodanese activity, but use autoc1aved soil
samples.

37-3.5 Debydrogenases

37-3.5.1 Introduction
Biological oxidation of organic compounds is generally a dehydroge-
nation process, and there are many dehydrogenases (enzymes catalyzing
dehydrogenation), which are highly specific. The overall process for dehy-
drogenation may be presented as follows:

XH2 + A~X + AH2 [24]

where XH2 is an organic compound (hydrogen donor) and A is a hydrogen

acceptor. The dehydrogenase enzyme systems apparently fulfill a signifi-
cant role in the oxidation of soil organic matter as they transfer H from
substrates to acceptors. Many different specific dehydrogenase systems are
involved in the dehydrogenase of soils; these systems are an integral part
of the microorganisms. Therefore, the result of the assay of dehydrogenase
activity would show the average activity of the active population (Skujins,
1976).
The most widely used method for determination of dehydrogenase
activity is that developed by Lenhard (1956). This method involves colo-
rimetric determination of 2,3,5-triphenyl formazan (TPF) produced by the
ENZYMES 821

reduction of 2,3,5-triphenyltetrazolium chloride (TIC) by soil microorgan-

isms. This assay has received considerable attention during the past decade
(Skujins, 1967; Klein et aI., 1971; Ross, 1971), presumably because it is
believed to provide a good index of microbial activity. But this belief seems
to be based entirely on Stevenson's (1959) report of a good correlation
between oxygen uptake and TIC dehydrogenase activity of some Cana-
dian soils.
Studies by Ross (1973) indicate that oxygen uptake and dehydroge-
nase activity are not closely related. Both activities are influenced by the
bacterial population and residual plant enzyme content and are negatively
correlated with catechol (phenolic) content. Work by Skujins (1973)
showed that dehydrogenase activity was highly correlated with CO 2 re-
lease, proteolytic activity, and nitrification potential. The highest activity
was in the top 3 cm of an arid soil, but again there was no correlation with
microbial numbers. Because the dehydrogenase activity depends on the
total metabolic activity of soil microorganisms, its value in different soils
containing different populations does not always reflect the total numbers
of viable microorganisms isolated on a particular medium (Skujins, 1976).
Dehydrogenase activity often is correlated with microbial respiration when
exogenous C sources are added to soils (Frankenberger & Dick, 1983).
Several treatments may affect dehydrogenase activity in soils. Toluene
and CHCl3 can strongly inhibit the activity of dehydrogenases in soils but
have little effect at low concentrations. Inhibition of up to 70% of the
original activity may occur in soils treated with 3% chloramphenicol. Be-
cause of this inhibition effect, bacteriostatic and bacteriocidal compounds
are included in the reaction mixture for assay of dehydrogenases in soils.
Thus, the contribution to the observed dehydrogenase activity by prolif-
erating microorganisms is unknown but can be anticipated to be substan-
tial, especially in assays involving soils amended with C substrates and
incubated for 24 h or more. Even in short-term assays, dehydrogenase
activity may be influenced not only by enzyme concentration but also by
the nature and concentration of added and endogenous C substrates and by
alternate electron acceptors (Ladd, 1978). Bremner and Tabatabai (1973)
have shown that added Fe203' Mn02' SO~-, POl-, and Cl- stimulated
soil dehydrogenase activity, whereas N0 3-, N0 2-, and Fe 3- seemed to
inhibit this activity. The apparent inhibition could be due to some of these
latter compounds acting as alternative electron acceptors.

37-3.5.2 Principles
Tetrazolium salts are representatives of a unique class of compounds.
These compounds have combinations of desirable properties. They are
quarternary NH/ salts and, as such, possess a high degree of water solu-
bility. The water solubility varies considerably and depends on the nature
and properties of the substituted groups. The solubility of TIC is suffi-
ciently great to allow the salt to be used in a water solution. This color-
less or pale-colored tetrazolium salt possess the property of being easily
822 TABATABAI

transformed into intensely colored, water-insoluble, methanol-soluble for-

mazan by reduction. The apparent redox potential of TIC is about -0.08
V, which makes this compound act as an acceptor for many dehydrogena-
ses (Mattson et aI., 1947).
The method described is based on extraction with methanol and col-
orimetric determination of the TPF produced from the reduction of TIC in
soils. The transformation occurs through a rupture of the ring, and the
general reaction is

o
[2S]

O~ -N ~+CI­
- I II
N N
~ /
C Triphenyl formazan

o
I

2,3, 5-Triphenyltetrazollium chloride

37-3.5.3 Assay Method (Casida et aI., 1964)

37-3.5.3.1 Special Apparatus
1. Test tubes, 16 by ISO mm.
2. Spectrophotometer or colorimeter.
37-3.5.3.2 Reagents
1. Calcium carbonate (CaC03 ), reagent grade.
2. 2,3,S-Triphenyltetrazolium chloride (TIC), 3%: Dissolve 3 g of
TIC (Calbiochem, Los Angeles) in about 80 mL of water, and
adjust the volume to 100 mL with water.
3. Methanol, analytical reagent grade.
4. Triphenyl formazan (TPF) standard solution: Dissolve 100 mg of
TPF (Calbiochem, Los Angeles) in about 80 mL of methanol, and
adjust the volume to 100 mL with methanol. Mix thoroughly.
37-3.5.3.3 Procedure. Thoroughly mix 20 g of air-dried soil ( < 2 mm)
and 0.2 g of CaC03 , and place 6 g of this mixture in each of three test tubes.
To each tube add 1 mL of 3% aqueous solution of TIC and 2.S mL of
distilled water. This amount of liquid should be sufficient that a small
amount of free liquid appears at the surface of the soil after mixing. Mix the
contents of each tube with a glass rod, and stopper the tube and incubate
it at 37°C. After 24 h, remove the stopper, add 10 of methanol, and
stopper the tube and shake it for 1 min. Unstopper the tube, and filter the
suspension through a glass funnel plugged with absorbent cotton, into a
100-mL volumetric flask. Wash the tube with methanol and quantitatively
transfer the soil to the funnel, then add additional methanol (in 1O-mL
portions) to the funnel until the reddish color has disappeared from the
ENZYMES 823

cotton plug. Dilute the filtrate to a 100-mL volume with methanol. Mea-
sure the intensity of the reddish color by using a spectrophotometer at a
wavelength of 485 nm and a 1-cm cuvette with methanol as a blank. Cal-
culate the amount of TPF produced by reference to a calibration graph
prepared from TPF standards. To prepare this graph, dilute 10 mL of TPF
standard solution to 100 mL with methanol (100 !!g of TPF mL ~ 1). Pipette
5-, 10-, 15-, and 20-mL aliquots of this solution into 100-mL volumetric
flasks (500, 1000, 1500, and 2000 !!g of TPF 100 mL ~1), make up the
volumes with methanol, and mix thoroughly. Measure the intensity of the
red color of TPF as described for the samples. Plot the absorbance readings
against the amount of TPF in the 100-mL standard solutions.

37-3.5.4 Comments
In general, tetrazolium salts are photosensitive, the gross change ob-
servable with visible light being the acquisition of a yellow color. Although
visible light causes yellowing, UV light of short-wave length brings about
reduction of TPF in aqueous solutions (Rust, 1955). The TPF produced is
also light sensitive. On long exposure to visible light, TPF undergoes a
color change from red to a yellow modification. Storage of the yellow
modification in the dark, however, reverses the reaction, and the color
gradually returns to its original red. The phenomenon is due to a cis-trans
isomerization.
It is important that the TPF produced be extracted quantitatively from
soils. This can be accomplished by washing the incubated soil in the funnel
with small increments of methanol. Care should be taken to prevent tur-
bidity in the methanol extract, because any turbidity would lead to higher
dehydrogenase activity values than those expected. Also, the procedure
described should be adhered to, because any changes in pH, temperature,
incubation time, substrate concentration, or amount of soil incubated will
lead to changes in the results obtained by this method. Also, toluene
severely inhibits the activity of this enzyme in soils (Frankenberger &
Johanson, 1986).
Standard curves prepared as described are reproducible, and it is not
necessary to prepare a calibration graph with each set of assay. The TPF
color produces is stable if stored in the dark, and none of the color changes
just described can be detected under normal laboratory conditions.

37-3.6 ~-Glucosidase

37-3.6.1 Introduction
The enzymes acting on glycosyl compounds (EC 3.2), including gly-
coside hydrolases (EC 3.2.1), have been among the hydrolases least stud-
ied in soils. The general name glycosidases or glycoside hydro lases has been
used to describe a group of enzymes that catalyze the hydrolysis of different
glycosides. The general equation of the reaction is
glycosides + H20 ~ sugar + aglycons. [26]
824 TABATABAI

In general, a glycoside may be defined as a mixed acetal resulting from

the exchange of an alkyl or aryl group for the H atom of the hemiacetal
hydroxyl group of a cyclic aldose or ketose. The aglycon (or genin) is the
noncarbohydrate portion attached to the carbohydrate moiety (glycosyl
residue) of the glycoside. The terms glycoside and glucoside have long been
used interchangeably. To avoid ambiguity, it is now customary to designate
as glycoside those substances that on acid hydrolysis liberate one or several
monosaccharides and an aglycon. The Commission of Enzymes of the
International Union of Biochemistry has classified all these enzymes into 39
groups (Florkin & Stotz, 1964). These include enzymes such as cellulase
and amylase and some important glycosidases that catalyze the hydrolysis
of disaccharides. Glycosidases have usually been named according to the
type of bond that they hydrolyze. Among the glycosidases, a-glucosidase
(obsolete name maltase, EC 3.2.1.20), which catalyzes the hydrolysis of
a-D-glucopyranosides, and ~-glucosidase (obsolete name gentiobiase or
cellobiase, EC 3.2.1.21), which catalyzes the hydrolysis of ~-D-gluco­
pyranosides, are involved in hydrolysis of maltose and cellobiose, respec-
tively. Other important glycosidases are a-galactosidase (obsolete name
melibiase, EC 3.2.1.22) and ~-galactosidase (obsolete name lactase, EC
3.2.1.23). These enzymes catalyze the hydrolysis of melibiose and lactose,
respectively. ~-Glucosidase activity is the most predominant of the four
enzymes in soils (Eivazi & Tabatabai, 1988, 1990).
Glucosidases and galactosidases are widely distributed in nature.
These enzymes have also been detected in soils (Skujins, 1967, 1976). The
wide distribution of ~-glucosidase in fungi (Jermyn, 1958), yeast (Barnett
et aI., 1956), and plants (Veibel, 1950) has made possible the study of this
enzyme. ~-Glucosidase is more dominant in soils than a-glucosidase and a-
and ~-galactosidases. The hydrolysis products of ~-glucosidases are be-
lieved to be important energy sources for microorganisms in soils.
Recently, Eivazi and Tabatabai (1988) surveyed the methods available
for assay of ~-glucosidase activity in soils and developed a simple method
for determination of the activity of this enzyme. These studies also showed
that this enzyme is inactivated in soils at 70°C and that its activity is
correlated with organic C content of surface soils and of soil profiles.
Kinetic studies indicated that the Km values of this enzyme for p-nitrophe-
nyl-~-D-glucoside (PNG) in surface soils range from 1.3 to 2.4 mM and that
the average activation energy value of ~-glucosidase in three soils was 28 kJ
mol-I.

37-3.6.2 Principles
The principles of the method described for assay of ~-glucosidase
activity are similar to those of assay of phosphodiesterase (section 37-
3.2.2). The method is based on colorimetric determination of the p-nitro-
phenol released by ~-glucosidase when soil is incubated with buffered (pH
6.0) PNG solution and toluene. The incubation is carried out at 37°C for
1 h. The p-nitrophenol released is extracted by filtration after addition of
ENZYMES 825

0.5 M CaCl2 and 0.1 M of THAM buffer pH 12. It is important to treat the
incubated soil sample with THAM buffer pH 12 instead of the 0.5 M of
NaOH used for extraction of p-nitrophenol in assay of phosphomo-
noesterases (section 37-3.2.2) and arylsulfatase (section 37-3.3.2), be-
cause the substrate of B-glucosidase, PNG is hydrolyzed with time in the
presence of excess NaOH. The CaCI2-THAM treatment described for ex-
traction of the p-nitrophenol released in the assay of B-glucosidase activity
serves the same purpose as that of CaCI2-NaOH used for extraction of this
enzymatic product in the assay of acid and alkaline phosphatases and ar-
ylsulfatase. The reaction involved is as follows:

[27]

When R = -----< >-- N0 2 '

the substrate is p-nitrophenyl-B-D-glucoside.

37-3.6.3 Assay Method (Eivazi & Tabatabai, 1988)

37-3.6.3.1 Special Apparatus
1. Incubation flasks, stoppers, incubator, and colorimeter or spectro-
photometer as described in section 37-3.2.3.1.1.
37-3.6.3.2 Reagents
1. Toluene, MUB pH 6.0, calcium chloride (CaCl2) (0.5 M), and
standard p-nitrophenol solution (described in section 37-
3.2.3.1.2).
2. p-Nitrophenyl-B-D-glucoside (PNG) solution, 0.05 M: Dissolve
0.654 g of PNG (Sigma Chemical Co., St. Louis, MO) in about 40
mL of MUB pH 6.0, and adjust the volume to 50 mL with the same
buffer. Store the solution in a refrigerator.
3. Tris(hydroxymethyl)aminomethane (THAM) buffers (0.1 M), pH
12 and ~ 10: Prepared as described in section 37-3.2.3.2.2.
37-3.6.3.3 Procedure. Place 1 g of soil ( < 2 mm) in a SO-mL Erlen-
meyer flask, add 0.25 mL of toluene, 4 mL of MUB pH 6.0, 1 mL of PNG
solution, and swirl the flask for a few seconds to mix the contents. Stopper
the flask, and place it in an incubator at 37°C. After 1 h, remove the
stopper, add 1 mL of 0.5 M CaCl2 and 4 mL of 0.1 M THAM buffer pH
826 TABATABAI

12, swirl the flask for a few seconds, and filter the soil suspension through
a Whatman no. 2v folded filter paper. Measure the intensity of the yellow
color of the filtrate with a Klett-Summerson photoelectric colorimeter, and
calculate the amount of p-nitrophenol released as described in section
37-3.2.3.1.3.
If the color intensity of the filtrate exceeds that of the highest p-ni-
trophenol standard solution, an aliquot of the filtrate should be diluted with
0.1 M THAM pH -10 until colorimeter reading falls within the limits of
the calibration graph.

37-3.6.4 Comments
Choice of buffer and buffer pH in the method described is based on
the finding by Eivazi and Tabatabai (1988) that the optimum pH of this
enzyme in soils is at MUB pH 6. The activities of a-glucosidase and a- and
~-galactosidase can be assayed by the procedure described using the cor-
responding substrates.
The PGN solution is stable for several days if stored in a refrigerator.
It is necessary to add CaCl2 to prevent dispersion of clay and extraction of
soil organic matter during treatment with THAM buffer pH 12 used for
extraction of the p-nitrophenol released.
Air-drying of field-moist soils results in marked increases in the activ-
ities of a- and ~-glucosidases and a- and ~-galactosidases in soils. The effect
of trace elements and inorganic salts on the activities of these enzymes vary
considerably among the elements, enzymes, and soils (Eivazi & Tabatabai,
1990).

37-3.7 Other Enzymes

The activities of several other enzymes have been detected in soils and
methods have been developed for their assays. These include methods for
measuring the activities of L-histidine ammonia-lyase (Frankenberger &
Johanson, 1982b,c), invertase (Frankenberger & Johanson, 1983a,b), and
nitrate reductase (Abdelmagid & Tabatabai, 1987; Fu & Tabatabai, 1989).
Other references for assay of enzyme activities in soils may be found in the
book edited by Burns (1978) and other references cited in this chapter.

REFERENCES
Abdelmagid, H.M., and M.A. Tabatabai. 1987. Nitrate reductase activity of soils. Soil BioI.
Biochem. 19:421-427.
Alberty, R.A. 1956. Enzyme kinetics. Adv. Enzymol. Relat. Subj. Biochem. 17:1-64.
Al-Khafaji, A.A., and M.A. Tabatabai. 1979. Effects of trace elements on arylsulfatase
activity in soils. Soil Sci. 127:129-133.
Allison, J.P., W.J. Mandy, and G.B. Kitto. 1971. The substrate specificity of L-asparaginase
from Alcaligenes eutrophus. FEBS Lett. 14:107-108.
Anderson, G. 1967. Nucleic acids, derivatives, and organic phosphates. p. 67-90. In A.D.
McLaren and G.H. Peterson (ed.) Soil biochemistry. Vol. 1. Marcel Dekker, New
York.
ENZYMES 827

Barnett, J.A., M. Ingram, and T. Swain. 1956. The use of ~-glucosidase in classifying yeast.
J. Gen. Microbiol. 15:529-555.
Beck, T., and H. Poschenrieder. 1963. Experiments on the effect of toluene on the soil
microflora. Plant Soil 18:346-357.
Berg, G.G., and L.H. Gordon. 1960. Presence oftrimetaphosphatase in the intestinal mucosa
and properties of the enzyme. J. Histochem. Cytochem. 8:85-91.
Bidwell, R.G.S. 1974. Plant physiology. p. 173-206. MacMillan, New York.
Blakeley, R.L., J.A. Hinds, H.E. Kunze, E.C. Webb, and B. Zerner. 1969. Jack bean urease
(EC 3.5.1.5). Determination of a carbamoyl-transfer reaction and inhibition by hydrox-
amic acids. Biochemistry 8:1991-2000.
Blanchar, R.W., and L.R. Hossner. 1969. Hydrolysis and sorption reactions of orthophos-
phate, pyrophosphate, tripolyphosphate, and trimetaphosphate anions added to an
Elliot soil. Soil Sci. Soc. Am. Proc. 33:141-144.
Brams, W.H., and A.D. McLaren. 1974. Phosphatase reactions in a column of soil. Soil BioI.
Biochem. 6:183-189.
Bray, H.G., S.P. James, LM. Raffan, B.E. Ryman, and W.V. Thorpe. 1949. The fate of
certain organic acids and amindes in the rabbit. Biochem. J. 44:618-625.
Bremner, J.M. 1955. Studies on soil humic acids: 1. The chemical nature of humic nitrogen.
J. Agric. Sci. 46:247-256.
Bremner, J.M., and R.L. Mulvaney. 1978. Urease activity in soils. p. 149-196. In R.G. Burns
(ed.) Soil enzymes. Academic Press, New York.
Bremner, J .M., and M.A. Tabatabai. 1973. Effect of some inorganic substances on TIC assay
of dehydrogenase activity in soils. Soil BioI. Biochem. 5:385-386.
Browman, M.G., and M.A. Tabatabai. 1978. Phosphodiesterase activity of soils. Soil Sci.
Soc. Am. J. 42:284-290.
Burns, R.G. 1978. Enzyme activity in soil: Some theoretical and practical considerations. p.
295-340. In R.G. Burns (ed.) Soil enzymes. Academic Press, New York.
Burns, R.G., A.H. Pukite, and A.D. McLaren. 1972. Concerning the location and persis-
tence of soil urease. Soil Sci. Soc. Am. Proc. 36:308-311.
Busman, L.M., and M.A. Tabatabai. 1984. Determination of trimetaphosphate added to
soils. Commun. Soil Sci. Plant Anal. 15:1257-1268.
Busman, L.M., and M.A. Tabatabai. 1985a. Hydrolysis of trimetaphosphate in soils. Soil Sci.
Soc. Am. J. 49:630-636.
Busman, L.M., and M.A. Tabatabai. 1985b. Factors affecting enzymic and nonenzymic
hydrolysis of trimetaphosphate in soils. Soil Sci. 140:421-428.
Campbell, H.A., L.T. Mashburn, E.A. Boyce, and L.J. Old. 1967. Two L-asparaginases from
Escherichia coli B: Their separation, purification, and antitumor activity. Biochemistry
6:721-730.
Cantarella H., and M.A. Tabatabai. 1983. Amides as sources of nitrogen for plants. Soil Sci.
Soc. Am. J. 47:599-603.
Casida, L.E., Jr., D.A. Klein, and T. Santoro. 1964. Soil dehydrogenase activity. Soil Sci.
98:371-376.
Cawse, P.A. 1975. Microbiology and biochemistry of irradiated soils. p. 213-267. In E.A.
Paul and A.D. McLaren (ed.) Soil biochemistry. Vol. 3. Marcel Dekker, New York.
Cedar, H., and J .H. Schwartz. 1967. Localization of the two L-asparaginases in anaerobically
grown Escherichia coli. J. BioI. Chern. 242:3753-3755.
Cervelli, S., P. Nannipieri, B. Ceccanti, and P. Sequi. 1973. Michaelis constant of soil acid
phosphatase. Soil BioI. Biochem. 5:841-845.
Chandramohan, D., K. Devendran, and R. Natarajan. 1974. Arylsulfatase activity in marine
sediments. Mar. BioI. 27:89-92.
Clarke, P.H. 1970. The aliphatic amidases of Pseudomonas aeruginosa. Adv. Microb. Physiol.
4:179-222.
Conrad, J.P. 1940a. Catalytic activity causing the hydrolysis of urea in soils as influenced by
several agronomic factors. Soil Sci. Soc. Am. Proc. 5:238-241.
Conrad, J.P. 1940b. Hydrolysis of urea in soils by thermolabile catalysis. Soil Sci. 49:253-263.
Conrad, J.P. 1940c. The nature of the catalyst causing the hydrolysis of urea in soils. Soil Sci.
50: 119-134.
Conrad, J.P. 1942a. The occurrence and origin of ureaselike activities in soils. Soil Sci.
54:367-380.
828 TABATABAI

Conrad, J.P. 1942b. Enzymatic vs. microbial concepts of urea hydrolysis in soils. Agron. J.
34:1102-1113.
Conrad, J.P. 1943. Some effects of developing alkalinities and other factors upon ureaselike
activities in soils. Soil Sci. Soc. Am. Proc. 8:171-174.
Cooper, P.J.M. 1972. Aryl sulphatase activity in Northern Nigerian soils. Soil BioI. Biochem.
4:333-337.
Cosgrove, D.J. 1967. Metabolism of organic phosphate in soil. p. 216-228. In A.D. McLaren
and G.H. Peterson (ed.) Soil biocheInlstry. Vol. 1. Marcel Dekker, New York.
Deng, S., and R.P. Dick. 1990. Sulfur oxidation and rhodanese activity in soils. Soil Sci.
150:552-560.
Dick, R.P., and S. Deng. 1991. Multivariate factor analysis of sulfur oxidation and rhodanese
activity in soils. Biogeochemistry 12:87-101.
Dick, R.P., and M.A. Tabatabai. 1986a. Hydrolysis of polyphosphates by com roots. Plant
Soil 94:247-256.
Dick, R.P., and M.A. Tabatabai. 1986b. Hydrolysis of polyphosphates in soils. Soil Sci.
142:132-140.
Dick, R.P., and M.A. Tabatabai. 1987. Factors affecting hydrolysis of polyphosphates in soils.
Soil Sci. 143:97-104.
Dick, W.A., N.G. Juma, and M.A. Tabatabai. 1983. Effects of soils on acid phosphatase and
inorganic pyrophosphatase of com roots. Soil Sci. 136:19-25.
Dick, W.A., and M.A. Tabatabai. 1977a. An alkaline oxidation method for determination of
total phosphorus in soils. Soil Sci. Soc. Am. J. 41:511-514.
Dick, W.A., and M.A. Tabatabai. 1977b. Determination of orthophosphate in aqueous so-
lutions containing labile organic and inorganic phosphorus compounds. J. Environ.
Qual. 6:82-85.
Dick, W.A., and M.A. Tabatabai. 1978a. Inorganic pyrophosphatase activity in soils. Soil
BioI. Biochem. 10:59-65.
Dick, W.A., and M.A. Tabatabai. 1978b. Hydrolysis of organic and inorganic phosphorus
compounds added to soils. Geoderma 21:175-182.
Dick, W.A., and M.A. Tabatabai. 1983. Activation of soil pyrophosphate by metal ions. Soil
BioI. Biochem. 15:359-363.
Dick. W.A., and M.A. Tabatabai. 1984. Kinetic parameters of phosphatases in soils and
organic waste materials. Soil Sci. 137:7-15.
Dick, W.A., and M.A. Tabatabai. 1987. Kinetics and activities of phosphates-clay complexes.
Soil Sci. 143:5-15.
Dick, W.A., and M.A. Tabatabai. 1992. Significance and potential uses of soil enzymes. p.
95-127. In F.B. Metting, Jr. (ed.) Soil microbial ecology: Application in agriculture and
environmental management. Marcel Dekker, New York.
Douglas, L.A., and J.M. Bremner. 1970a. Extraction and colorimetric determination of urea
in soils. Soil Sci. Soc. Am. Proc. 34:859-862.
Douglas, L.A., and J.M. Bremner. 1970b. Colorimetric determination of microgram quan-
tities of urea. Anal. Lett. 3:79-87.
Douglas, L.A., and J.M. Bremner. 1971. A rapid method of evaluating different compounds
as inhibitors of urease activity in soils. Soil BioI. Biochem. 3:309-315.
Douglas, L.A., H. Sochtig, and W. Flaig. 1978. Colorimetric determination of urea in soil
extracts using an automated system. Soil Sci. Soc. Am. J. 42:291-292.
Dowd, J.E., and D.S. Riggs. 1965. A comparison of estimates of Michaelis-Menten kinetic
constants from various linear transformations. J. BioI. Chern. 240:863-869.
Drobni'K, J. 1956. Degradation of asparagine by the soil enzyme complex. Cesk. Mikrobiol.
1L47.
Dunn, C. G., W.L. Campbell, H. Fram, and A. Hutchins. 1948. Biological and photochemical
effect of high energy, electrostatically produced roentgen rays and cathode rays. J.
Appl. Phys. 19:605-616.
Eivazi, F., and M.A. Tabatabai. 1977. Phosphatases in soils. Soil BioI. Biochem. 9:167-172.
Eivazi, F., and M.A. Tabatabai. 1988. Glucosidases and galactosidases in soils. Soil BioI.
Biochem.20:601-606.
Eivazi, F., and M.A. Tabatabai. 1990. Factors affecting glucosidase and galactosidase activ-
ities in soils. Soil BioI. Biochem. 22:891-897.
ENZYMES 829

Eremenko, V.V., A.V. Zhukov, and A.Y. Nikolaev. 1975. Asparaginase and glutaminase
activity of Pseudomonas fluorescens during continuous culturing. Microbiology 44:
550-555.
Fearon, W.R. 1939. The carbamido diacetyl reaction: A test for citruline. Biochem. J.
33:902-907.
Feder, J. 1973. The phosphatases. p. 475-508. In E.J. Griffith et al. (ed.) Environmental
phosphorus handbook. John Wiley and Sons, New York.
Fiorkin, M., and E.H. Stotz. 1964. p. 126-134. In Comprehensive biochemistry. Vol. 13.
Elsevier North-Holland, New York.
Frankenberger, W.T., Jr., and W.A. Dick. 1983. Relationships between enzyme activities and
microbial growth and activity indices in soil. Soil Sci. Soc. Am. J. 47:945-951.
Frankenberger, W.T., Jr., and J .B. Johanson. 1982a. Effect of pH on enzyme stability in soils.
Soil BioI. Biochem. 14:433-437.
Frankenberger, W.T., Jr., and J.B. Johanson. 1982b. L-Histidine ammonia-lyase activity in
soil. Soil Sci. Soc. Am. J. 46:943-948.
Frankenberger, W.T., Jr., and J.B. Johanson. 1982c. Distribution of L-histidine ammonia-
lyase activity in soils. Soil Sc. 136:347-353.
Frankenberger, W.T., Jr., and J.B. Johanson. 1983a. Method of measuring invertase activity
in soils. Plant Soil 74:301-311.
Frankenberger, W.T., Jr., and J.B. Johanson. 1983b. Factor affecting invertase activity in
soils. Plant Soil 74:313-323.
Frankenberger, W.T., Jr., and J.B. Johanson. 1986. Use of plasmolytic agents and antiseptics
in soil enzyme assays. Soil BioI. Biochem. 18:209-213.
Frankenberger, W.T., Jr., and M.A. Tabatabai. 1980a. Amidase activity in soils: I. Methods
of assay. Soil Sci. Soc. Am. J. 44:282-287.
Frankenberger, W.T., Jr., and M.A. Tabatabai. 1980b. Amidase activity in soils: II. Kinetic
parameters. Soil Sci. Soc. Am. J. 44:532-536.
Frankenberger, W.T. Jr., and M.A. Tabatabai. 1981a. Amidase activity in soils: III. Stability
and distribution. Soil Sci. Soc. Am. J. 45:333-338.
Frankenberger, W.T., Jr., and M.A. Tabatabai. 1981b. Amidase activity in soils: IV. Effects
of trace elements and pesticides. Soil Sci. Soc. Am. J. 45:1120-1124.
Frankenberger, W.T., Jr., and M.A. Tabatabai. 1982. Amidase and urease activities in plants.
Plant Soil 64:153-166.
Frankenberger, W.T., Jr., and M.A. Tabatabai. 1985. Characteristics of an amidase isolated
from a soil bacterium. Soil BioI. Biochem. 17:303-308.
Frankenberger, W.T., Jr., and M.A. Tabatabai. 1991a. L-Asparaginase activity of soils. BioI.
Fert. Soils 11 :6-12.
Frankenberger, W.T., Jr., and M.A. Tabatabai. 1991b. Factors affecting L-asparaginase ac-
tivity in soils. BioI. Fert. Soils 11:1-5.
Frankenberger, W.T., Jr., and M.A. Tabatabai. 1991c. L-Glutaminase activity of soils. Soil
BioI. Biochem. 23:869-874.
Frankenberger, W.T., Jr., and M.A. Tabatabai. 1991d. Factors affecting L-glutaminase ac-
tivity in soils. Soil BioI. Biochem. 23:875-879.
Freney, J.R. 1961. Some observations on the nature of organic sulphur compounds in soils.
Aust. J. Agric. Res. 12:424-432.
Fromageot, C. 1950. Sulfatases. p. 517-526. In J.B. Sumner and K. Myrback (ed.) The
enzymes. Vol. 1. Part 1. Academic Press, New York.
Fu, M.H., and M.A. Tabatabai. 1989. Nitrate reductase activity in soils: Effect of trace
elements. Soil BioI. Biochem. 21:943-946.
Galstyan, A.S., and E.G. Saakyan. 1973. Determination of soil glutaminase activity. Dokl.
Akad. Nauk SSSR 209:1201-1202.
Gilliam, J .W., and E.C. Sample. 1968. Hydrolysis of pyrophosphate in soils: pH and biologi-
cal effects. Soil Sci. 106:352-357.
Gorin, G. 1959. On the mechanisms of urease action. Biochim. Biophys. Acta 34:268-270.
Halstead, R.L. 1964. Phosphatase activity of soil as influenced by lime and other treatments.
Can. J. Soil Sci. 44:137-144.
Hashimoto, I., J.D. Hughes, and O.D. Philen, Jr. 1969. Reaction of triammonium pyro-
phosphate with soils and soil minerals. Soil Sci. Soc. Am. Proc. 33:401-405.
Hossner, L.R., and D.P. Phillips. 1971. Pyrophosphate hydrolysis in flooded soil. Soil Sci.
Soc. Am. Proc. 35:379-383.
830 TABATABAI

Houghton, C., and EA. Rose. 1976. Liberation of sulphate from sulphate esters by soils.
Appl. Environ. Microbiol. 31:969-976.
Hynes, M.J. 1970. Induction and repression of amidase enzymes in Aspergillus nidulans. J.
Bacteriol. 103:482-487.
Hynes, M.J. 1975. Amide utilization in Aspergillus nidulans: Evidence for a third amidase
enzyme. J. Gen. Microbiol. 91:99-109.
Imada, A., S. Igarasi, K. Nakahama, and M. Isono. 1973. Asparaginase and glutaminase
activities of microorganisms. J. Gen. Microbiol. 76:85-80.
Irving, G.C., and D.J. Cosgrove. 1976; The kinetics of soil acid phosphatase. Soil BioI.
Biochem. 8:335-340.
Ivey, EJ., and K. Shaver. 1977. Enzymic hydrolysis of polyphosphate in the gastrointestinal
tract. J. Agric. Food Chern. 25:128-130.
Jakoby, W.B., and J. Fredericks. 1964. Reactions catalyzed by amidases. Acetamidase. J.
BioI. Chern. 239:1978-1982.
Jermyn, M.A. 1958. Fungal cellulases. Aust. J. BioI. Sci. 11:114-126.
Joshi, J.G., and P. Handler. 1962. Purification and properties of nicotinamidase from Torula
cremoris. J. BioI. Chern. 237:929-935.
Juma, N.G., and M.A. Tabatabai. 1977. Effects of trace elements on phosphatase activity in
soils. Soil Sci. Soc. Am. J. 41:343-346.
Juma, N.G., and M.A Tabatabai. 1978. Distribution of phosphomonoesterases in soils. Soil
Sci. 126:101-108.
Juma, N.G., and M.A Tabatabai. 1988a. Hydrolysis of organic phosphates by com and
soybean roots. Plant Soil 107:31-38.
Juma, N.G., and M.A. Tabatabai. 1988b. Phosphatase activity in com and soybean roots:
Conditions for assay and effects of metals. Plant Soil 107:39-47.
Juma, N.G., and M.A. Tabatabai. 1988c. Comparison of kinetic and thermodynamic param-
eters of p.hosphomonoesterases of soils and of com and soybean roots. Soil BioI. Bio-
chern. 20:533-539.
Kelly, M., and P.H. Clarke. 1962. An inducible amidase produced by a strain of Pseudomonas
aeruginosa. J. Gen. Microbiol. 27:305-316.
Kiss, S., M. Dracan-Bularda, and D. Radulescu. 1975. Biological significance of enzymes
accumulated in soil. Adv. Agron. 27:25-87.
Klein, D.A., T.e. Loh, and R.L. Goulding. 1971. A rapid procedure to evaluate the dehy-
drogenase activity of soils low in organic matter. Soil BioI. Biochem. 3:385-387.
Kornberg, S.R. 1956. Tripolyphosphate and trimetaphosphate in yeast extracts. J. BioI.
Chern. 218:23-31.
Kowalenko, C.G., and L.E. Lowe. 1975. Mineralization of sulfur from four soils and its
relationships to soil carbon, nitrogen, and phosphorus. Can. J. Soil Sci. 55:9-14.
Kramer, M. 1957. Phosphatase-Enzym-Aktivitlit als Anzeiger des biologisch nutzbaren phos-
phors im Boden. Naturwissenschaften 44:13.
Kramer, M., and G. Yerdei. 1959. Application of the method of phosphatase activity deter-
mination in agricultural chemistry. Sov. Soil Sci. (English translation) 9:1100-1103.
Kroll, L., and M. Kramer. 1955. Der Einfluss der Tonmineralien auf die Enzym-Aktivitlit der
Bodenphosphatase. Naturwissenschaften 42: 157 -158.
Kuprevich, Y.F., and T.A Shcherbakova. 1971. Comparative enzymatic activity in diverse
types of soil. p. 167-201. In AD. McLaren and J.J. Skujins (ed.) Soil biochemistry.
Vol. 2. Marcel Dekker, New York.
Ladd, J.N. 1978. Origin and range of enzymes in soil. p. 51-96. In R.G. Bums (ed.) Soil
enzymes. Academic Press, New York.
Lai, C.M., and M.A Tabatabai. 1992. Kinetic parameters of immobilized urease. Soil BioI.
Biochem. 24:225-228.
Lang, K. 1933. Die rhodanbildung im tierkorper. Biochem. Z. 259:243-256.
Lenhard, G. 1956. Die Dehydrogenaseaktivitlit des Bodens als Mass flir die Mikroorganis-
mentlitigkeit im Boden. Z. Pflanzenernaehr. Dueng. Bodenkd. 73:1-11.
Mattenheimer, H. 1956. Die Sybstratspezifitat "anorganischer" Polyund Metaphosphatasen:
III. Papier-chromatographische Vnter suchungen beim enzymatischen Abbau von an-
organischen Polyund Metaphosphaten. Hoppe-Seyler; s. Z. physiol. Chern. 303: 125-138.
Mattson, A.M., C.O. Jensen, and R.A. Dutcher. 1947. Triphenyl-tetrazolium chloride as a
dye for vital tissue. Science 106:294-295.
ENZYMES 831

McLaren, A.D. 1969. Radiation as a technique in soil biology and biochemistry. Soil BioI.
Biochem. 1:63-73. ,
McLaren, A.D. 1975. Soil as a system of humus and clay immobilized enzymes. Chern. Scr.
8:97-99.
McLaren, A.D., and E. Packer. 1970. Some aspects of enzyme reactions in heterogeneous
systems. Adv. Enzymol. Relat. Subj. Biochem. 33:245-308.
McLaren, A.D., L. Reshetko, and W. Huber. 1957. Sterilization of soil by irradiation with an
electron beam, and some observations on soil enzyme activity. Soil Sci. 83:497-502.
Meyerhof, 0., R. Shatas, and A. Kaplan. 1953. Heat of hydrolysis of trimetaphosphate.
Biochim. Biophys. Acta 12:121-127.
Michaelis, L., and M.L. Menten. 1913. Die Kinetik der Invertinwirtung. Biochem. Z. 49:333-
369.
Neilands, J.B., and P.K. Stumpf. 1964. Outlines of enzyme chemistry. 2nd ed. John Wiley
and Sons, New York. p. 57-159.
Neptune, A.M.L., M.A. Tabatabai, and J.J. Hanway. 1975. Sulfur fractions and carbon-
nitrogen-sulfu~-phosphorus relationships in some Brazilian and Iowa soil. Soil Sci. Soc.
Am. Proc. 39.51-5,.
Nicholls, R.G., and A.R. Roy. 1971. Arylsulfatases. p. 21-41. In P.D. Boyer (ed.) The
enzymes. Vol. 5. 3rd ed. Academic Press, New York.
Nor, Y.M., and M.A. Tabatabai. 1975 Colorimetric determination of microgram quantities of
thiosulfate and tetrathionate. Anal. Lett. 8:537-547.
Nor, Y.M., and M.A. Tabatabai. 1976. Extraction and colorimetric determination of thio-
sulfate and tetrathionate in soils. Soil Sci. 122:171-178.
Nor, Y.M., and M.A. Tabatabai. 1977. Oxidation of elemental sulfur in soils. Soil Sci. Soc.
Am. J. 41:736-741.
Paul, E.A., and A.D. McLaren. 1975. Biochemistry of the soil subsystem. p. 1-36. In E.A.
Paul and A.D. McLaren (ed.) Soil biochemistry. Vol. 3. Marcel Dekker, New York.
Paulson, K.N., and L.T. Kurtz. 1969. Locus of urease activity in soil. Soil Sci. Soc. Am. Proc.
33:897-901.
Pearson, R.W., A.G. Norman, and C. Ho. 1941. The mineralization of the organic phos-
phorus of various compounds in soils. Soil Sci. Soc. Am. Proc. 6:168-175.
Pettit, N.M., L.J. Gregory, R.B. Freedman, and R.G. Bums. 1977. Differential stabilities of
soil enzymes: Assay and properties of phosphatase and arylsulphatase. Biochim. Bio-
phys. Acta 485:357-366.
Pettit, N.M., A.R.J. Smith, R.B. Freedman, and R.G. Bums. 1976. Soil urease: Activity,
stability and kinetic properties. Soil BioI. Biochem. 8:479-484.
Pierpoint, W.S. 1957. The phosphatase and metaphosphatase activities of pea extracts. Bio-
chern. J. 65:67-76.
Porter, L.K. 1965. Enzymes. p. 1536-1549. In C.A. Black et al. (ed.) Methods of soil
analysis. Part 2. Agron. Monogr. 9. ASA, Madison, WI.
Privat de Garilhe, M. 1967. Enzymes in nucleic acid research. Holden-Day, San Francisco. p.
259-278.
Pulford, I.D., and M.A. Tabatabai. 1988. Effect of waterlogging on enzyme activities in soils.
Soil BioI. Biochem. 20:215-219.
Quastel, J.H. 1946. Soil metabolism. The Royal Institute of Chemistry of Great Britain and
Ireland, London.
Ramirez-Martinez, J.R. 1968. Organic phosphorus mineralization and phosphatase activity
in soils. Folia Microbiol., Praha 13:161-174.
Ramirez-Martinez, J.R., and A.D. McLaren. 1966a. Determination of phosphatase activity
by a fluorimetric technique. Enzymologia 30:243-253.
Ramirez-Martinez, J.R., and A.D. McLaren. 1966b. Some factors influencing the determi-
nation of phosphatase activity in native soils and in soils sterilized by irradiation. En-
zymologia 31:23-38.
Ray, R.C., N. Behera, and n. Sethunathan. 1985. Rhodanese activity of flooded and non-
flooded soils. Soil BioI. Biochem. 17:159-162.
Razzell, W.E., and H.G. Korana. 1959. Studies on polynucleotides: III. Enzymatic degra-
dation. Substrate specificity and properties of snake venom phosphodiesterase. J. BioI.
Chern. 234:2105-2113.
Reiner, J.M. 1959. Behavior of enzyme systems. Burgess Publ. Co., Minneapolis.
832 TABATABAI

Reithel, F.J. 1971. Ureases. p. 1-21. In P.D. Boyer (ed.) The enzymes. Vol. 4. Academic
Press, New York.
Roberge, M.R. 1970. Behavior of urease added to unsterilized, steam-sterilized and gamma
radiation-sterilized black spruce humus. Can. J. Microbiol. 16:865-870.
Roberge, M.R. 1978. Methodology of soil enzyme measurement and extraction. p. 341-370.
In R.G. Burns (ed.) Soil enzymes. Academic Press, New York.
Roberts, J., J.S. Holcenber, and W.C. Dolowy. 1972. Isolation, crystallization, and proper-
ties of Achromobacteraceae, glutaminase-asparaginase with antitumor activity. J. BioI.
Chern. 247:84-90.
Rogers, H.T. 1942. Dephosphorylation of organic phosphorus compounds by soil catalysts.
Soil Sci. 54:439-446.
Ross, D.J. 1971. Some factors influencing the estimation of dehydrogenase activities of some
soils under pasture. Soil BioI. Biochem. 3:97-110.
Ross, D.J. 1973. Some enzyme and respiratory activities of tropical soils from New Hebrides.
Soil BioI. Biochem. 5:559-567.
Rotini, O.T. 1935. La transformazione enzimatica deJl'-urea nell terreno. Ann Labor. Ric.
Ferm. Spallanzani 3:143-154.
Rotini, O.T., and L. Carloni. 1953. La transformazione die metafosfati in ortofasfati pro-
mossa dal terreno Agrario. Ann. Spero Argrar. 7:1789-1799.
Roy, A.B. 1960. The synthesis and hydrolysis of sulfate esters. Adv. Enzymol. 22:205-235.
Rust, J.B. 1955. Chemical properties of the tetrazolium salts. Ann. N.Y. Acad. Sci. 17:379-
384.
Sayre, F. W., and E. Roberts. 1958. Preparation and some properties of a phosphate-activated
glutaminase from kidneys. J. BioI. Chern. 233:1128-1134.
Schmidt, G., and M. Laskowski, Sr. 1961. Phosphate ester cleavage (Survey). p. 3-35. In
P.D. Boyer et al. (ed.) The enzymes. 2nd ed. Academic Press, New York.
Searle, P.L., and W.T Speir. 1976. An automated colorimetric method for the determination
of urease activity In soil and plant material. Commun. Soil Sci. Plant Anal. 7:365-374.
Segel, I.H. 1975. Enzyme kinetics. John Wiley and Sons, New York.
Sequi, P. 1974. Enzymes in soil. Ital. Agric. 111:91-109.
Singh, B.B., and M.A. Tabatabai. 1978. Factors affecting rhodanese activity in soils. Soil Sci.
125:337-342.
Skujins, J. 1967. Enzymes in soil. p. 371-414. In A.D. McLaren and O.H. Peterson (ed.) Soil
biochemistry. Vol. 1. Marcel Dekker, New York.
Skujins, J. 1973. Dehydrogenase: An indicator of biological activities in arid soils. Bull. Ecol.
Res. Commun. NFR 17:235-241.
Skujins, J. 1976. Extracellular enzymes in soil. CRC Crit. Rev. Microbiol. 4:383-421.
Skujins, J. 1978. History of abiontic soil enzyme research. p. 1-49. In R.G. Burns (ed.) Soil
enzymes. Academic Press, New York.
Skujins,oJ.J., L. Braal, and A.D. McLaren. 1962. Characterization of phosphatase in a
terrestrial soil sterilized with an electron beam. Enzymologia 25:125-133.
Skujins, J.J., and A.D. McLaren. 1968. Persistence of enzymatic activities in stored and
geologically preserved soils. Enzymologia 34:213-225.
Skujins, J.J., and A.D. McLaren. 1969. Assay of urease activity using 14C-urea in stored,
geologically preserved and an irradiated soils. Soil BioI. Biochem. 1:89-99.
Sowden, F.J. 1958. The forms of nitrogen in the organic matter of different horizons of soil
profiles. Can. J. Soil Sci. 38:147-154.
Speir, T.W. 1977. Studies on a climosequence of soils in tussock grasslands 10. Distribution
of urease, phosphatase and sulphatase activities in soil fractions. N. Z. J. Sci. 20:
151-157.
Speir, T.W., and D.J. Ross. 1975. Effects of storage on the activities of protease, urease,
phosphatase and sulphatase in three soils under pasture. N. Z. J. SCl. 18:231-237.
Speir, T.W., and D.J. Ross. 1978. Soil phosphatase and sulphatase. p. 197-250. In R.G.
Burns (ed.) Soil enzymes. Acadeffilc Press, New York.
Spencer, B. 1958. Studies on sulphatases: 20. Enzymic cleavage of arylhydrogen sulphates in
the presence of H2 180. BlOchem. J. 69:155-159.
Stevenson, I.L. 1959. Dehydrogenase activity in soils. Can. J. Microbiol. 5:229-235.
Stojanovic, B.J. 1959. Hydrolysis of urea in soil as affected by season and by added urease.
Soil Sci. 88:251-255.
ENZYMES 833

Stossel, P., G. Lazarovits, and E. W.B. Ward. 1981. Cytochemical staining and in vitro activity
of acid trimetaphosphatase in etiolated soybean hypocotyls. Can. J. Bot. 59:1501-1508.
Stott, D.E., W.A. Dick, and M.A. Tabatabai. 1985. Inhibition of pyrophosphatase activity in
soils by trace elements. Soil Sci. 139:112-117.
Sumner, J.B. 1951. Urease, p. 873-892. In J.B. Sumner and K. Myrblick (ed.) The enzymes.
Vol. 1. Part 2. Academic Press, New York.
Tabatabai, M.A. 1973. Michaelis constants of urease in soils and soil fractions. Soil Sci. Soc.
Am. Proc. 37:707-710.
Tabatabai, M.A. 1977. Effects of trace elements on urease activity in soils. Soil BioI. Bio-
chem. 9:9-13.
Tabatabai, M.A., and J .M. Bremner. 1969. Use of p-nitrophenyl phosphate for assay of soil
phosphatase activity. Soil BioI. Biochem. 1:301-307.
Tabatabai, M.A., and J.M. Bremner. 1970a. Arylsulfatase activity of soils. Soil Sci. Soc. Am.
Proc. 34:225-229.
Tabatabai, M.A., and J.M. Bremner. 1970b. Factors affecting soil arylsulfatase activity. Soil
Sci. Soc. Am. Proc. 34:427-429.
Tabatabai, M.A., and J.M. Bremner. 1971. Michaelis constants of soil enzymes. Soil BioI.
Biochem. 3:317-323.
Tabatabai, M.A., and J.M. Bremner. 1972a. Assay of urease activity in soils. Soil BioI.
Biochem.4:479-487.
Tabatabai, M.A., and J.M. Bremner. 1972b. Forms of sulfur, and carbon, nitrogen, and
sulfur relationships in Iowa soils. Soil Sci. 114:380-386.
Tabatabai, M.A., and W.A. Dick. 1979. Distribution and stability of pyrophosphatase in
soils. Soil BioI. Biochem. 11:655-659.
Tabatabai, M.A., and M.H. Fu. 1992. Extraction of enzymes from soils. p. 197-227. In G.
Stotzky and J-M. Bollag (ed.) Soil biochemistry. Vol. 7. Marcel Dekker, New York.
Tabatabai, M.A., and B.B. Singh. 1976. Rhodanese activity of soils. Soil Sci. Soc. Am. J.
40:381-385.
Tabatabai, M.A., and B.B. Singh. 1979. Kinetic parameters of the rhodanese reaction in
soils. Soil BioI. Biochem. 11:9-12.
Thornton, J.I., and A.D. McLaren. 1975. Enzymatic characterization of soil evidence. J.
Forensic Sci. 20:674-692.
Tower, D.B., E.L. Peters, and W.C. Curtis. 1963. Guinea pig serum L-asparaginase. J. BioI.
Chem. 238:983-993.
Tyler, G. 1974. Heavy metal pollution and soil enzymatic activity. Plant Soil 41:303-311.
Tyler, G. 1976a. Influence of vanadium on soil phosphatase activity. J. Environ. Qual.
5:216-217.
Tyler, G. 1976b. Heavy metal pollution, phosphatase activity and mineralization of organic
phosphorus in forest soils. Soil BioI. Biochem. 8:327-332.
Van Wazer, J.R. 1958. Phosphorus and its compounds. Vol. 1. Chemistry. Interscience Publ.,
New York.
Varner, J.E. 1960. Urease. p. 247-256. In P.D. Boyer et al. (ed.) The enzymes. Vol. 4.
Academic Press, New York.
Veibel, S. 1950. ~-Glucosidase. p. 583-620. In J.B. Sumner and K. Myrblick (ed.) The
enzymes. Vol. 1. Part 1. Academic Press, New York.
Wall, M.C., and K.J. Laidler. 1953. The molecular kinetics of the urea-urease system-IV.
The reaction in an inert buffer. Arch. Biochem. Biophys. 43:299-306.
White, A., P. Handler, and E.L. Smith. 1968. Principles of biochemistry. 4th ed. McGraw-
Hill Publ., New York. p. 223-246.
Wriston, J.C., Jr. 1971. L-Asparaginase. p. 101-121. In P.D. Boyer (ed.) The enzymes. Vol.
4. Academic Press, New York.
Yashphe, J. 1973. Estimation of micro amounts of urea and carbamyl derivatives. Anal.
Biochem.52:143-153.
Zantua, M.I., and J .M. Bremner. 1975a. Comparison of methods of assaying urease activity
in soils. Soil BioI. Biochem. 7:291-295.
Zantua, M.I., and J.M. Bremner. 1975b. Preservation of soil samples for assay of urease
activity. Soil BioI. Biochem. 7:297-299.
Zantua, M.I., and J .M. Bremner. 1976. Production and persistence of urease activity in soils.
Soil BioI. Biochem. 8:369-374.