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ANAl.

YTICAL

BIOCHEMISTRY

Steady-State

C. NICK

109,

Kinetic

261-265

(1980)

Studies of Arginase with an Improved


Spectrophotometric
Assay

PACE, ANDRES

BUONANNO,

Received
A direct spectrophotometric
t 1967. At~crl. Biochem.
18, 102)
substrate
concentrations,
for
kinetic studies of the enzyme.
reported
and compared
with
number of problems
encountered

AND

was supported
by Grant
Institutes
of Health.

SIMMONS-HANSEN

July 7. 1980

assay for arginase developed


by R. L. Ward and P. A. Srere
is extended
so that it can be used at pH 7.5 or 9.5. at higher
assaying
crude sources of arginase.
and for steady-state
Steady-state
kinetic studies of beef and rat liver arginase are
Gmilar
results obtained
using other assay procedures.
A
in steady-state
kinetic studies of argina\e
are discussed.

Arginase (t-arginine amidinohydrolase,


EC 3.5.3. I) catalyzes the cleavage of arginine
to ornithine and urea. A surprisingly large
number of techniques have been developed
for assaying arginase (see ( 1) and references
therein). The most popular of these measures
urea formation and requires a 60-min boiling
step (2). In addition, the product is light
labile and requires special precautions during
boiling and cooling (3). This inconvenience
coupled with the fact that arginase generally
requires prior activation by heating (4) result
in a tedious assay of limited precision. The
wide range of Michaelis constants reported
for rat and beef liver arginase (0.3 to over
20 mM). and the diversity of conclusions
about the role of Mn+ in the activation of
the enzyme are, in part, reflections of the
assays available. In 1967, Ward and Srere
(5) described a direct, spectrophotometric
assay for arginase based on the difference
in absorption between the products and reactants at 205.7 nm. This approach is much
simpler and faster than the indirect assays
and has since been used by several investigators (6-10). We have extended this
method so that it can be used at higher
This research
from the National

JEANNIE

Direct

AM 191 12-02

substrate concentrations, for assaying crude


samples of arginase, and at either pH 7.5 of
9.5. We show that the method can be used
for steady-state kinetic studies of the enzyme
and report Michaelis constants determined
for the rat and beef liver enzymes under
various conditions.
MATERIALS

AND METHODS

Partially purified beef liver arginase (33.3


unitsimg) was purchased from Worthington
Biochemical Company (Lot AR36C869).
Schimkes procedure ( 11) was used for
purifying rat liver arginase. The specific
activity of the final product ranged from
650 to 1050 unitsimg in different purifications. Enzymes and chemicals for the
coupled-enzyme assay were purchased
from Boehringer-Mannheim. For the data in
Fig. I. the A grade hydrochlorides of arginine
and ornithine from Calbiochem. and ultrapure urea from SchwarziMann were used.
All other chemicals were the best grade
available from Sigma Chemical Company.
The absorption spectra and some of the
initial velocities were measured with a
Cary Model 15 spectrophotometer. Other
initial velocities were recorded with a Gilford Model 250 spectrophotometer. The

262

PACE.

BUONANNO.

Wavelength
FIG.
and
and

I.

Absorption

spectra

for

urea at pH 9.5. E = Molar


k = tArC ~ Ed,,,, - t,,,-, .,,.

arginine.

absorption

ornithine.
coefficient.

Archibald
method (2) and the coupledenzyme method ( 12) were used for measuring some of the initial velocities in Fig. 2.
For the experiments in Fig. 2 and in Table
2. the arginase was dialyzed against 0.05 or
5 IIIM MnCl,, 10 mM Tris-HCl,
pH 7. for
4 to 6 h at 4C and then activated by heating
for 1 hat 37C. Identical results are obtained
when the heating step is omitted. The reaction is initiated by adding 0.1 ml of the
arginase to 2.5 ml of a substrate solution
at either pH 7.5 or 9.5: consequently,
the
MP concentrations
will be only 0.0019 or
0.19 mM when the velocity is measured.
RESULTS
The absorption
spectra
of arginine,
ornithine, and urea at pH 9.5 are shown in
Fig. 1. The difference absorption coefficient,
Ae = E.~,, - Ed,,, ~ t,,,,;,, is also shown and
can be seen to increase sharply below 215
nm. Consequently,
the absorbance change
accompanying the conversion of arginine to

AND

HANSEN

ornithine plus urea will be maximized by


using the lowest possible wavelength. At pH
7.5 similar results are obtained but AC and
the molar absorption coefficients for arginine
and ornithine are reduced. The presence
of I mM Mn does not affect the he values
at pH 7.5. Molar absorption coefficients for
arginine and the k values at pH 7.5 and
9.5 are listed in Table I.
In a typical assay of arginase. we use 2.5
ml of an I -arginine solution with a concentration in the range from 0. I to I5 KIM.
Arginase (0.5 to 3.5 units) is added to this
solution to initiate the reaction and the
absorbance
is recorded.
The rate of absorbance
change can be determined
by
drawing a tangent to the progress curve at
the beginning of the reaction. Initial velocities
in terms of concentrations
can then be
calculated from these rates using the ALE
values in Table 1. With care, velocities
can be determined with a precision of better
than +5% over a wide range of substrate
concentrations.
Excellent agreement is obtained when different wavelengths
are used
for measuring the velocities. A wavelength
IABLk
MOLAR
ANI)

ABSoRPrlON
DII I t RFN(~
,I

CO1.l I 1c1kh
ABSORPTION
pH
pH

9.5

.NI)

15 I OR AR(~INI~I
Corl~t
IC II VI\
7.5

7.5

pH

Y.5

Wavelength
(nm)
215
214
213
217
211
110
7OY

42
hl

I34
177

85
123
I74
136
344
477

74 I
Y71
I x4

205
104

I At

7)
103

2 35
314
423
564

108
107
106

hf-

Art

643
X63
I I41
1478

IhI5
cm
t\rc

.
t<t,,,

t,,n 8

tl

At

340

YX

27x
334

I I7
155

396
47)
SY3
734

IYY
Xl
352
46X
60

YI
II40
1471

X16
I Oh2

I754
7193

1361
17%

ARGINASE

263

ASSAY

c
:
I
E

0.5

0.2

0.1

v/S

0.3

Imln-I

FIG. 2. Eadie-Hofstee
plots for the hydrolysis
of L-arginine
by beef liver arginase.
Initial velocities
determined
by Archibald
assay at pH 7.0. 37C (0): by the direct assay at pH 9.5. 25T (13): or by the
coupled-enzyme
assay at pH 9.5. 37C (a).

must be selected which gives an absorbance


in the usable range of the spectrophotometer
(generally less than 3.0) when the substrate
and enzyme are mixed. The data in Table 1
can be used to calculate the absorbance of
the substrate solution; the absorbance due
to the enzyme solution depends on the source
of the enzyme and must be determined
independently.
As pointed out by Ward and Srere (5).
this assay can be used with crude samples
of arginase and gives results in excellent
agreement with the Archibald procedure (2)
when used to monitor the purification of
arginase. It is common practice to maintain a high Mn+ concentration
(50 mM)
during the purification of arginase 14). This
creates a problem with the direct assay not
previously noted. Since the pH optimum for
arginase is near pH 9.5, the enzyme is generally assayed by adding a small aliquot of the
enzyme solution at pH 7-7.5 to a solution
of the substrate at pH 9.5. If 50 mM Mn+ is
present in the enzyme solution, there will be
a sizable increase in the absorbance resulting from the exposure of Mn2+ to pH 9.5.
This absorbance change will diminish the
decrease in absorbance due to the hydrolysis

of arginine and lead to erroneous results.


This problem can be avoided by assaying
the arginase at pH 7.5. or minimized
by
maintaining the Mn+ concentration of the
arginase solution at 5 mM or less.
The direct assay is particularly convenient
for obtaining initial velocities for steadystate kinetic studies of arginase. Some
results obtained using the direct assay are
compared with results obtained using the
Archibald method (2) and the coupledenzyme assay ( 12) in Eadie-Hofstee
plots in
Fig. 2. The data from the direct assay yield
a Michaelis constant of 1.3 mM, while
values of 1.6 and 1.4 mM are obtained from
the Archibald and coupled-enzyme assays.
respectively.
We have also used the direct assay to
determine
the Michaelis
constants for
L-arginine with purified rat liver arginase.
Initial velocities were measured at substrate
concentrations ranging from 0.15 to 5.0 mM
and the Michaelis constant and standard
errors were estimated as described by Wilkinson (13). Corrections were made for substrate depletion (14) and corrections for
product inhibition
were shown to be insignificant. The results are presented in

264

PACE,
TABLE

THE

MICHAELIS
L-ARGININE

BUONANNO.

CoNsrAN
r COR THY HYDROLYSIS
BY RAI
LIVER
ARGINASI

ok

K,,, (mhil
M/
(mM)

pH 7.5

pH 9.5

0.05
.s

I.55 ? 0.16
1.33 t 0.13

1.03 + 0.19
1.13 -t 0.13

Km values
Wilkinson
(13).

? SE

estimated

as

described

by

Table 2. For reasons discussed


below,
estimates of K,,, obtained using the direct
assay are generally lower than estimates
obtained using other assays.
DISCUSSION
Michaelis constants ranging from 0.3 ( 10)
to 20 mM (15) have been reported for the
hydrolysis
of arginine by beef and rat liver
arginase. Several factors which may contribute to this poor agreement are considered below.
The evidence is good that both beef and
rat liver arginase bind 1 mol of Mn
per subunit (7.16) and that the enzyme can
be activated by heating in the presence of a
high concentration of Mn + ( IZ- 17). typically
5 min at 55C in the presence of 50 mM
Mn)+ (4). It is less certain, however,
that
the resulting enzyme resembles arginase as
it exists in \lit*o. The total concentration
of Mn+ in rat liver is approximately
0.024
mM ( I .3 pgig liver) (18) and the free Mn+
concentration,
considerably less (19). Thus,
distinctly
nonphysiological
conditions
are
used to activate arginase and there are
substantial discrepancies
among published
results. (Compare, for example, the results
in ( 16) and (20) for rat liver arginase.) Furthermore. high Mn
concentrations
interfere
with both the direct (see Results) and Archibald (21) assays. Finally. Mn+ binds to
zwitterionic
arginine with an association
constant of 335 Mm (22) and can thereby

AND

HANSEN

reduce the free substrate concentration.


For
these reasons, we recommend
the use of
lower Mn+ concentrations
and lower temperatures
for the activation
of arginase.
The results in Table 2 show that very similar
results are obtained when arginase is activated at 37C in the presence of 0.05 or
5 mM Mn+.
The pH optima for beef and rat liver
arginase are between pH 9.0 and 10.5 ( 15).
and the enzymes have been assayed at
several different pH values in this range.
This complicates kinetic studies of arginase.
The pk of the cu-amino group of arginine
is 9.04 (23) or 9.36 (22). Consequently,
the
relative concentration
of monocationic and
zwitterionic
arginine will vary depending
on the pH used for the assay and it is not
established with certainty which is the better
substrate (24). In addition. the degree of
complex
formation
between
Mn? and
arginine will increase with increasing pH.
and, finally. Mn+ solutions
turn brown
when exposed to high pH. Thus, even though
the activity
of arginase is lower,
much
more consistent
results can be obtained
when kinetic studies are carried out at
physiological
pH.
Failure to correct for substrate depletion
can substantially
increase the K,,, value
determined from a steady-state kinetic study
(14). For example,
in a typical kinetic
study of arginase using the Archibald assay
and a 5-min incubation of substrate and
enzyme, calculations
show that the K,,,
would be over 25% greater if no correction
were made for substrate depletion. Since 5and IO-min incubations are commonly used
in kinetic studies of arginase, this factor
probably contributes to the wide range ofk,,,
values observed.
A related problem is the substrate concentration range employed in a steady-state
kinetic study. Cleland recommends a range
of substrate concentrations
from 0.2 to 5
K,,, for determining K,,, (25). Rosenfeld cf trl.
( 10) report K,,, = 0.3-0.4 mM for beef liver
arginase but use only a 0.5 to 1.O mM range

ARCINASE

of arginine

concentrations.

Hirsch-Kolb

rt (11. (15). on the other hand, report K,,,

values of 7 and 20 mM for beef and rat


liver arginase, respectively, and use a substrate concentration
range from 5 to 100
mM arginine. The use of an inappropriate
range of substrate concentration is likely to
contribute to errors in the K,,, values.
The values of K,,, we report are in best
agreement with values of 1.0 mM for beef
(7). 2.4 mM for rat (26), and 1.4 tItM for
rabbit (27) liver arginase. Most of the k,,,
values reported for liver arginases are considerably higher than these values, probably,
in part, for reasons discussed above.
In summary, the information
presented
here will allow arginase to be assayed under
a wider range of conditions and should
prove useful for steady-state kinetic studies
of the enzyme.
REFERENCES
I. Ruegg.
U. T.. and Russell.
A. S. (1980) And/.
Eiochem.
102, 206-212.
2. Archibald.
R. M. (1945) J. Biol. Ch~nr. 157, 507517.
3. Van Slyke.
D. D.. and Archibald,
R. M. (1946)
J. Bid. Chem. 165, 293-309.
4. Schimke.
R. T. (1970) in Methods
Enzymology
(Tabor.
H., and Tabor. C. W.. eds.). Vol. l7A,
pp. 313-317.
Academic
Press, New York.
5. Ward, R. L.. and Srere. P. A. (1967) ,4no/. Riothem.
18, IOZ- 106.
6. Hirsch-Kolb.
H.. and Greenberg.
D. M. (1968)
J. Bic~f. C-hem. 243, 6123-6129.
7. Harell,
D.. and Sokolovsky.
M. (1972) Eur. J.
Bioc~hrm.
25, 102-108.

ASSAY

365

8. Kaysen.
G. A., and Strecker,
H. J. (1973) Bioc~hertl. J. 133, 779-788.
9. Kuchel.
P. W.. Nichol,
L. W.. and Jeffrey.
P. D.
(1975) J. Bird. Chenz. 2.50, 8222-8227.
10. Rosenfeld.
.I. L.. Dutta.
S. P., Chheda.
G. B.,
and Tritsch,
G. L. (1975) Bioc,him.
Bioph.vt.
Ac,rc~ 410, 164- 166.
I I. Schimke,
R. T. (1964) .!. Rio/. Ckcm.
239, 380%
3817.
I?. Bergmeyer,
H. U. (1974) in Methods
of Enzymatic
Analysis,
Vol. 4. pp. 1794-1798.
Academic
Press, New York.
13. Wilkinson,
G. N. (1961) Biochern.
J. 80, 324-332.
14. Pace. C. N. (1980) Trends Eiochrnr.
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IS. Hirsch-Kolb,
H., Heine. J. P., Kolb, H. J.. and
Greenberg,
D. M. t 1970) Con~p.
Bioc,hem.
Phy.tio/.
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16. Hirsch-Kolb.
H.. Kolb,
H. J.. and Greenberg.
D. M. (197l)J.
Viol. Chcnr. 246, 395-401.
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J. W. (1966) Camp. Biochcm.
Physird.
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18. Thiers.
R. E., and Vallee.
B. L. (1957) J. Bid.
Chern. 226, 91 l-920.
19. Scrutton,
M. C.. Utter. M. F.. and Mildvan.
A. S.
(1966) J. Bird. Chenr. 241, 3480-3487.
20. Musznska.
G., and Ber. E. (1979). frrr. .I. Hi+
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M.. Niina.
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I. (1976)
Rioinorgnn.
Chern. 6. l33- 143.
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E. R.. and Mat-tell, A. E. (1970) .I. Inrq.
Nut,/. Chrm.
32, 91 l-926.
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E. J.. and Edsall. J. T. (1943) Proteins,
Amino
Acids,
and Peptides,
p. 85, Hafner,
New York.
34. Greenberg.
D. M. (1960) in The Enzymes
(Bayer,
P. D., Lardy,
H.. and Myrback.
K.. eds.). 2nd
rd..
Vol. 4. pp. 257-367.
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Press.
New York.
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26. Schimke.
R. T. ( 196?).1. Viol. Clren). 237,459-468.
27. Vielle-Breitburd.
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Chrrn. 247, 1327- 13.5.