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.
Applications
L-Arginase causes the following reaction:
The quarternary structure of native rat liver arginase has been described by Hirsch-Kolb, H & Greenberg, D.M.,” Molecular characterisitics of rat liver arginase”. 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.
Reagents
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 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.
Procedure
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 (delta)E546nm of sample and (delta)E546nm of standard.
