Carboxypeptidase Y Assay

Method: The assay measures the rate of leucine liberated during the enzymatic hydrolysis of benzyloxycarbonyl-L-phenylalanyl-L-leucine under the specified conditions. One unit hydrolyzes 1 u mole of benzyl-oxycarbonyl-L-phenylalanyl-L-leucine per minute at 25°C, pH 6.5 under the conditions specified.

Reagents

• Substrate: 1 mM Benzyloxycarbonyl-L-phenylalanyl-L-leucine in 50 mM sodium phosphate, 0.15 M Sodium chloride, pH 6.5. Note: 0.5 ml of DMSO (dimethyl solfoxide) is used to dissolve the benzyloxycarbonyl-L-phenylalanyl-L-leucine before mixing with the buffer.
• 0.050 M sodium phosphate, 0.15 M Sodium chloride, pH 6.5
• 4% Ninhydrin in Methyl Cellosolve
• 0.2 M Sodium citrate, 7.1 mM stannous chloride, pH 5.0
• Ninhydrin-citric acid mixture. Prepare by mixing 50 ml each of 4% ninhydrin in methyl cellosolve and 0.2 M sodium citrate (pH 5.0) - 7.1 mM stannous chloride. Stir for 15 minutes.
• 50% (V/V) n-Propanol
• 0.05 M L-leucine

Leucine Standard Curve

Pipette the following amounts into test tubes and mix well:

0.05 M NaPO4 0.15 M NaCl (pH 6.5)	0.050 M L-Leucine	Concentratation (uM L-leucine)
4.90 ml	200 μl	0.392
5.00 ml	100 μl	0.196
5.05 ml	50 μl	0.098
5.08 ml	20 μl	0.039
5.10 ml	0.0 μl	0

Dilute each of the above 1:5 with NaPO₄ buffer.

Enzyme

Allow enzyme to sit at room temperature for 2-3 hours before assaying. This equilibration stabilizes activity. Prepare a 1 mg/ml solution of the enzyme, using reagent grade water. Dilute to concentrations of 5, 10, 15 and 20 μg/ml by using 5, 10, 15 and 20 μl of the 1 mg/ml solution, and taking to a final volume of 1 ml with reagent grade water.

Procedure

Pipette 1.0 ml of the substrate solution into test tubes labeled 5, 10, 15, 20 and BLANK. Pipette 1.0 ml of the 1:5 L-leucine into test tubes labeled 0.392, 0.196, 0.098, 0.039, and LEUCINE BLANK. Pre-incubate the five test tubes containing the substrate solutions for 10 minutes at 25°C. Start the enzyme reaction by pipetting 50 μl of the four enzyme samples into their respective test tubes. Add 50 μl of reagent grade water to the tube labeled BLANK. Allow to react at 25°C for 10 minutes. Add 1.0 ml of the ninhydrin reagent to each of the 10 test tubes. Place all tubes in a boiling water bath for 15 minutes. Remove tubes from bath and cool to below 30°C. Add 5.0ml of the 50% propanol solution to each of the test tubes and mix well. Read the optical density of all tubes at 570 nm.

Calculation: