Arginase Assay

Method: A number of assay procedures have been used (Greenberg 1960). More recently, Geyer and Dabich (1971) have 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.