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L-Arginase

(L-Arginine amidinohydrolase; EC 3.5.3.1)

L-Arginase causes the following reaction:

               L-Arginase
L-arginine + H2O ------> L-ornithine + urea

The quarternary structure of native rat liver arginase has been described by Hirsch-Kolb, H & Greenberg, D.M.," Molecular characterisitics of rat liver arginase", J. Biol Chem., 243, 6123-6129 and Baranczyk-Kuzma A., Porembska Z. & Mochnacka, I., Acta Biochim Polon, 23, 151-163. EDTA treatment dissociated the enzyme into inactive subunits of 30,000 daltons each. Addition of Mn2+ ions restored the activity and caused reassociation of subunits to the natve form of 120,000 daltons.

1.Source:	Bovine Liver
Form:	Freeze-dried
Solubility:	Soluble in water
Stability:	-20° C; -4° F
Activity (approx.):	100 U/mg protein
Protein (approx.):	95%
Catalog No.:	182A0100

$3.00/KU

One unit of enzyme activity is defined as that amount of enzyme that causes the hydrolysis of one micromole of L-arginine per minute at 37°C and pH 9.5.

0.2 M L-arginine solution: 1.05 g L-arginine monochloride/25 ml, adjusted to pH 9.5 with 1 N NaOH.
12.5 mM Urea standard sol'n. (75 mg urea/100 ml)
0.084 N Sulfuric acid (2.32 ml conc. H₂SO₄/1000 ml)
0.3 M Sodium tungstate, pH 7.0: 10 g Na₂WO₄●2H₂O/100 ml, pH adjusted to 7.0 with 1 N H₂SO₄
0.03 M Tungstic acid solution: Mix 9 parts H₂SO₄ (3) with 1 part sodium tungstate solution (4). Prepare fresh prior to assay.
60% (v/v) Phosphoric acid (60 ml conc. H₃PO₄, approx. 85-87%/100 ml).
60 mM Diacetylmonoxime/3.3 mM thiosemicarbazide reagent: Mix 600 mg diacetylmonoxime + thiosemicarbazide/100ml. Mix 10 parts H₃PO₄ (6) with 2 parts solution immediately prior to assay.
10 mM Manganese-maleate buffer: 10 mM Mn²⁺, 10 mM maleate (116 mg maleic acid anhydride/100 ml,, adjusted to pH 9.7 with 0.1 N NaOH; add 0.5 ml 2 M MnSO₄ solution and adjust to pH 7.5 with 0.1 M H₂SO₄.
L-Arginase: 1 mg /ml solution in 10 mM Manganese-maleate buffer (8) diluted to 1:500 dilution.

Set up water bath at 37°C.

Into two test tubes, pipette the following reagents:

	STANDARD	  SAMPLE
Arginine sol'n (1)        0.20 ml        0.20 ml
Distilled water                          0.20 ml
Urea standard sol'n. (2)  0.20 ml
Diluted enzyme sol'n (9)                 0.10 ml

Incubate at 37°C for 30 minutes.

Then add the following:

Tungstic acid solution (5)         4.50 ml         4.50 ml
Diluted enzyme sol'n (9)           0.10 ml

Allow to stand for 5 min. at room temperature and centrifuge the precipitate and add 0.2 ml of supernatant and 5 ml of Reagent 7 to develop the color to standard and blank. Heat in boiling water for 30 min. and measure the E_546nm of sample and E_546nm of standard.

Activity (U/mg) =	(ΔE_546nm/min)(2.5)
	(ΔE_546nm/min)(30)(0.1)(mg Enz./ml)

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