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(L-Arginine amidinohydrolase; EC

L-Arginase causes the following reaction:

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 

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 37C and pH 9.5.

  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●2H2O/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 37C.
  2. Into two test tubes, pipette the following reagents:
    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 37C 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 (delta)E546nm of sample and (delta)E546nm of standard.

Activity (U/mg) = (ΔE546nm/min)(2.5)
(ΔE546nm/min)(30)(0.1)(mg Enz./ml)

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