This procedure may be used for determination of Endoproteinase Glu-C enzymatic activity using N-t-Boc-L-glutamic acid α-phenyl ester as the substrate.
This continuous UV spectrophotometric rate determination (A270, Light path = 1 cm) is based on the following reaction:
Unit Definition: One unit will hydrolyze 1.0 µmole of N-t-Boc-L-glutamic acid α-phenyl ester per minute at pH 7.8 at 37°C. One unit is equivalent to ~0.004 casein digestion unit.
Use ultrapure water (≥18 MΩx cm resistivity at 25°C) for the preparation of reagents.
Acid solution (5 N Phosphoric Acid) – Prepare a 0.11 mL/mL solution of 85 wt. % phosphoric acid, Product Number 695017, in ultrapure water.
Buffer solution (200 mM Tris-Phosphate Buffer, pH 7.8 at 37°C) – Prepare 2.5-fold dilution of stock 0.5 M Trizma Base buffer, prepared using Product Number T1503, using ultrapure water. Adjust to pH 7.8 at 37°C with Acid.
Substrate solution (60 mM N-t-Boc-L-glutamic acid a-phenyl ester) – Prepare 19.4 mg/mL solution of N-t-BOC-GAPE, using Bachem Product Number A-4540 in 1,4-Dioxane, Product Number 34857.
Note: Prepare Fresh. Keep at Room Temperature.
Test solution (Endoproteinase Glu-C Enzyme Solution) – Immediately before use, prepare a solution containing 0.5-2.0 units/mL of Endoproteinase Glu-C, in purified water.
Final Assay Conditions – In a 3.0 mL reaction mix, the final concentrations are 190 mM Tris, 1 mM N-tert-Butoxy-Carbonyl-L-Glutamic Acid α-Phenyl Ester, 1.7% 1,4-dioxane, and 0.01–0.2 units of Endoproteinase Glu-C.
1. Pipette (in mL) the following into suitable quartz cuvettes:
2. Mix by inversion and equilibrate using a suitably thermostatted spectrophotometer at 37°C. Then add (in mL):
3. Immediately mix by inversion and record the increase in A270 for ~5 minutes. Obtain the ΔA270/minute using the maximum linear rate for all the Tests and Blank using a minimum of 4 data points over a one minute time interval.
3.0 = Total Volume (in mL) of assay reaction mixture
df = Dilution factor
1.5 = Millimolar extinction coefficient of phenol at 270 nm
0.10 = Volume (in mL) of enzyme used