ANALYTICAL BIOCHEMISTRY 18, l&106 (1967)

A New Spectrophotometric Arginase Assay1

RAYMOND L. WARD AND PAUL A. SRERE2

Lawrence Radiation Laboratory, University 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)) cata- lyzes 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 orni- thine 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, orni- thine, and urea.

Each assay cuvet contains 2.5 ml of arginine, 2 X 10m3 M, 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 Energy Commission.

f Present address : Veterans Administration Hospital, Dallas, Texas. 102

ARGIXASB ASSAY 103

FIG. 1.

ornithine, and urea

,Ll 1850 I900 1950 2000 zu3v ZIUU

WAVELENGTH (ii,

Absorption spectra of equimolar concentrations (2 X 1WM) at pH 9.4.

S- e- 7-

TIME (min)

OSJ- 1 ,

A, 0 2 4 6 8 IO 12

TIME (min)

of argininc,

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 AND SRERE

0 20 40 60 60 100 I20 140 /q PROTEIN ADDED

FIG. 3. Initial velocity of absorbancy change as a function of enzyme concentration.

the linearity of the semilog plot (inset, Fig. 2) of initial arginine con- centration (a,,) divided by initial arginine concentration minus amount 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 8 IO

TIMEfmin)

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.

ARGINASE ASSAI 105

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 Greenberg (4) .

0 Colorimetrlc assay

n This ossoy

Roholt and

0 2 4 6 8 IO I2 14 TIME (min)

FIQ. 5. Comparison of amount of urea produced during a given reaction time period for a particular arginase activity as measured by a calorimetric assay and by this assay. The amount of urea formed in the spectrophotometric assay was assumed to be equivalent to the arginine cleaved.

We have measured simultaneously under identical conditions the formation of urea by a calorimetric procedure (2, 5) and the disappear- ance 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 for his technical assistance throughout the experiments. This work was supported in part by the Bio-Medica Division of the Lawrence Radiation Laboratorv.

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).