Arginase Assay

Method: Geyer and Dabich (1971) reported on an assay for arginase in tissue homogenates and Nishibe and Makino (1971) an automated method for erythrocyte enzyme. Hirsch-Kolb and Greenberg (1970) describe a microassay. The method used by this laboratory is based upon the colorimetric determination of released urea nitrogen, using 2,3-butanedione reagent. One unit releases one micromole of urea per minute at 37°C and pH 9.5 under the specified conditions.

Reagents

• 0.05 M Maleic acid, pH 7.0 with 0.05 M manganous sulfate
• 0.713 M L-arginine, pH 9.5
• Reagent grade water, pH 9.5 (20 mg NaOH per liter)
• 0.075%, 2,3-Butanedione in buffered arsenic-sulfuric acid

Enzyme

Incubate a one mg/ml solution of the enzyme in maleic-manganous sulfate buffer at 37°C for 4 hours. Following activation, dilute to 1-2 micrograms/ml in reagent grade water, pH 9.5.

Procedure

Pipette the following into screw capped tubes:

	Tube
	Blank	# 1	# 2
Water, pH 9.5	0.3 ml	0.1 ml	0.2 ml
Diluted, activated enzyme	----	0.2 ml	0.1 ml
Incubate in 37°C water bath for 5 minutes to achieve temperature equilibration. At timed intervals start reaction by adding:
Arginine	0.2 ml	0.2 ml	0.2 ml
Incubate for 30 minutes at 37°C. Stop reaction at timed intervals by adding:
B.U.N. Reagent	4.0 ml	4.0 ml	4.0 ml

Cap tubes and develop color by boiling in water bath for 12 minutes. Chill tubes for 3 minutes in an ice bath. Read the color at 490 nm against blank.

Calculation

Determine the micromoles of urea released from a standard urea curve in the range of 0.1-1.0 micromoles urea.