Leucine Aminopeptidase Assay

Method: Based on that of Mitz and Schlueter (1958), the hydrolysis of the peptide bond of leucinamide is measured spectrophotometrically at 238 nm. One unit of enzyme activity is equal to one micromole of L-Leucinamide hydrolyzed per minute at 25°C and pH 8.5.

Reagents

• 0.025 M Manganese chloride
• 0.125 M Magnesium chloride
• 0.5 M Tris⋅HCl buffer, pH 8.5
• 0.0625 M L-Leucine, pH 8.5. Note: The extinction coefficient of L-leucinamide should be checked upon each use by the technique outlined in the procedure.

Enzyme

Prior to assay, the enzyme must be activated for two hours at 37°C in the following:

0.025 M Manganese Chloride	0.1 ml
0.5 M Tris⋅HCl buffer	0.1 ml
Enzyme	*
Reagent grade water	q.s. to 2.3 ml

* The volume of the sample activated may vary, depending upon the material to be assayed; however, no more than 0.4 mg of the purified product should be added to the activation mixture.

Procedure

Determination of extinction coefficient

Prepare cuvettes as follows:

	Control	Test
0.0625 M L-leucine	2.0 ml	-----
0.125 M L-leucinamide	-----	1.0 ml
0.5 M Tris⋅HCl pH 8.0	0.1 ml	0.1 ml
0.125 M Magnesium chloride	0.1 ml	0.1 ml
Reagent grade water	0.2 ml	1.2 ml

Using the control as an absorbance blank, determine A₂₃₈ for test cuvette.

Calculate extinction coefficient (E_m) as follows:

Determination of reaction rates:

Incubate test cuvettes in the spectrophotometer at 25°C for 4-5 minutes to achieve temperature equilibration and establish a blank rate, if any. Add 0.1 ml of enzyme activation mixture and record decrease in A₂₃₈ from the linear portion of the curve. Some lag may occur during the initial period and should not be used in the calculation.

Calculation