L-Arginase causes the following reaction:

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

1. 0.2 M L-arginine solution: 1.05 g L-arginine monochloride/25 ml, adjusted to pH 9.5 with 1 N NaOH.
2. 12.5 mM Urea standard sol'n. (75 mg urea/100 ml)
3. 0.084 N Sulfuric acid (2.32 ml conc. H2SO4/1000 ml)
4. 0.3 M Sodium tungstate, pH 7.0: 10 g Na2WO4?H2O/100 ml, pH adjusted to 7.0 with 1 N H2SO4
5. 0.03 M Tungstic acid solution: Mix 9 parts H2SO4 (3) with 1 part sodium tungstate solution (4). Prepare fresh prior to assay.
6. 60% (v/v) Phosphoric acid (60 ml conc. H3PO4, approx. 85-87%/100 ml).
7. 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.
8. 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.
9. L-Arginase: 1 mg /ml solution in 10 mM Manganese-maleate buffer (8) diluted to 1:500 dilution.

1. Set up water bath at 37°C.
2. 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
   

3. Incubate at 37°C for 30 minutes.
4. Then add the following:

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

5. 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 E546nm of sample and E546nm of standard.