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ANALYTICAL

18, l&106

BIOCHEMISTRY

A New

Spectrophotometric

RAYMOND
Lawrence

Radiation

(1967)

L. WARD

Laboratory,

Arginase
AND

University

PAUL

Assay1

A. SRERE2

of California,

Livermore,

California

Received May 6, 1966

We wish to report a new method for the assay of arginase (L-arginine
amidinohydrolase,
EC 3.5.3.1).
Arginase, the terminal enzyme of the urea-ornithine
cycle (1)) catalyzes the cleavage of arginine to ornithine and urea. Present assays for
arginase determine the released urea either by a calorimetric procedure
(2) or by a manometric procedure with urease (3). Our new assay ‘is a
spectrophotometric
method based on the fact that the absorbancy of
arginase below 21OOA is larger than the combined absorbancies of ornithine and urea. A cleavage of arginine catalyzed by the enzyme thus
results in a net decrease in absorbancy at these wavelengths, allowing a
rapid and accurate assay for arginase activity.
METHODS

AND RESULTS

All spectral measurements have been made with a Cary model 14
recording spectrophotometer.
The ultraviolet
absorption spectra for
arginine, ornithine and urea are shown in Figure 1. We have arbitrarily
chosen to assay the enzyme at 2057 K where the molar absorptivities are
1.35 X lo3 for arginine, 0.36 X lo3 for ornithine, and 0.02 X lo3 for urea.
These absorptivities give an e value of 0.97 X lo3 for the disappearance
of arginine. For all assays we have used a fixed slit of 0.8 mm. Each
instrument should be standardized with known solutions of arginine, ornithine, and urea.
Each assay cuvet contains 2.5 ml of arginine, 2 X 10m3M, pH 9.4, and
the reaction is started by the addition of 0.125 ml of arginase. The
reference cuvet contains 2.5 ml of water and 0.125 ml of the same enzyme
solution. The absorption of light by arginine is partially balanced by
the insertion of a “screen” of known absorbance in the reference beam.
The change in absorbance at 2057A with time after an addition of
arginase is shown in Figure 2. This is a first-order reaction, as shown by
‘This work was performed under the auspices of the U. S. Atomic
Commission.
f Present address : Veterans Administration
Hospital, Dallas, Texas.
102

Energy

ARGIXASB

103

ASSAY

,Ll
1850

I900

1950

zu3v

2000

WAVELENGTH
FIG.

1.

ornithine,

Absorption spectra of equimolar
and urea at pH 9.4.

ZIUU

(ii,

concentrations

(2 X 1WM)

of argininc,

S-

e7-

TIME

(min)

1
,

OSJ-

A,

0

2

4

6
TIME

8

IO

12

(min)

FIQ. 2. Absorbancy aa a function of time after addition of arginase to an arginine
solution. Insert: First-order semilog plot of initial arginine concentration divided by
initial arginine concentration minus amount reacted at any time t.

104

WARD

0
FIG.

3.

20

AND

40

60
/q

SRERE

60
PROTEIN

100
ADDED

I20

140

Initial velocity of absorbancy change as a function of enzyme concentration.

the linearity

of the semilog plot

(inset, Fig. 2) of initial arginine conarginine concentration minus amount

centration (a,,) divided by initial
reacted at any time (2) versus time.
The rate of change of absorbance decreases with time; we have used
the rate observed in the first 30 set as the initial velocity of reaction.
Figure 3 is a plot of initial velocity versus enzyme concentration.

$3
3
5
,”

t

0

2

4

6
TIMEfmin)

8

IO

FIG. 4. Plot amount of arginine cleaved VB. time for a fixed arginaee activity and
at various arginine concentrations. Arginine disappearance wae estimated by the
decrease in absorbawe at 2057 k.

105

ARGINASE ASSAI

Since K, for arginine in the arginase reaction has been shown to be
high, it is not practical in this assay to use saturating concentrations of
arginine. The apparent velocity at 2.0 mM, the concentration used here,
is about one-third the maximum velocity of the reaction.
The assay can also be used for the quantitative estimat,ion of arginine;
at 0.8, 1.2, and 1.6 mM the reaction is complete in 10 min (Fig. 4). We
have in addition determined the pH-activity
relation using this assay,
with results essentially the same as those reported by Roholt and
Greenberg (4) .

0

2

4

0

Colorimetrlc

n

This

6
TIME

FIQ. 5. Comparison
period for a particular
this assay. The amount
to be equivalent to the

assay

ossoy

8

IO

I2

14

(min)

of amount of urea produced during a given reaction time
arginase activity as measured by a calorimetric assay and by
of urea formed in the spectrophotometric
assay was assumed
arginine cleaved.

We have measured simultaneously
under identical conditions the
formation of urea by a calorimetric procedure (2, 5) and the disappearance of arginine by the spectrophotometric
procedure described here.
These results (Fig. 5) indicate satisfactory agreement between the two
methods. We have used our assay also to follow the purification
of
arginase from rat liver and have found it to be very convenient. No
difficulty was encountered even in assaying initial extracts of this tissue.
dCKNOWLEDGMENT
The authors wish to thank Mr. Joseph Gonyeau
throughout the experiments. This work was supported
Division of the Lawrence Radiation Laboratorv.

for his technical assistance
in part by the Bio-Medica

106

WARD

AND

SRERE

REFERENCES
1. KREBS, H. A., AND HENSELEIT, K., 2. Physiol. Chem. 210,33 (1932).
2. ARCHIBALD, R. M., J. Biol. Chem. 157, 507 (1945).
3. BACH, S. J., AND &LIP,
J. D., B&him. Biophys. Acta 29,273 (1958).
4. ROHOLT, 0. A., JR., AND GREENBERG, D. M. Arch. Biochem. Biophys. 62,454 (1956)
5. VAN SLYICE, D. D., AND ARCHIBALD, R. M., .I. Biol. Chem. 165, 293 (1946).