Carboxypeptidase B1/CPB1 Protein, Mouse (HEK293, His)

Materials
Activation buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
Experimental buffer: 25 mM Tris, 0.1 M NaCl, pH 7.5
Carboxypeptidase B1/CPB1 Protein, Mouse (HEK293, His) (HY-P72869)
Trypsin
AEBSF, prepared as a 100 mM stock solution in DMSO
Substrate: Hippuryl-Arg, prepared as a 12.5 mM stock solution in deionized water
Standard: Hippuric acid (HY-W016562)
96-well transparent UV plate

Procedure
1. Preparation of Standards: Prepare hippuric acid dilutions at concentrations of 2000 μM, 1000 μM, 500 μM, 250 μM, 125 μM, 62.5 μM, 31.25 μM, 15.6 μM, 7.8 μM, and 3.9 μM. Perform triplicate wells for each concentration gradient.
2. Dilute trypsin to 500 µg/mL in activation buffer. Use this mixture to dilute the target protein to 100 µg/mL for activating Mouse CPB1.
3. Incubate at room temperature for 30 minutes.
4. Add AEBSF to a final concentration of 1 mM to stop the reaction.
5. Dilute the activated Mouse CPB1 to 2 µg/mL using assay buffer.
6. Dilute the substrate to 2 mM using assay buffer.
7. In a 96-well transparent UV plate, add 50 µL of 2 µg/mL Mouse CPB1. Include a substrate blank containing 50 µL assay buffer as a blank control. Perform triplicate wells for each gradient.
8. Add 50 µL of 2 mM substrate to all wells and initiate the reaction.
9. Read absorbance at 254 nm for 5 minutes in kinetic mode to obtain absorbance values.
10. Calculate specific activity: