Attention:

Certain features of Sigma-Aldrich.com will be down for maintenance the evening of Friday August 18th starting at 8:00 pm CDT until Saturday August 19th at 12:01 pm CDT.   Please note that you still have telephone and email access to our local offices. We apologize for any inconvenience.

Enzymatic Assay of Endoproteinase Glu-C (EC 3.4.21.19)

Description

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.

Reagents and Equipment Required

Phosphoric acid, Product Number 695017
Trizma® Base, Product Number T1503
N-t
-BOC-GAPE, Bachem Product Number A-4540
1,4-Dioxane, Product Number 34857

Precautions
Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Preparation Instructions

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.

Procedure

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:

Solution

Blank

Test 1

Test 2

Test 3

Buffer

2.85

2.85

2.85

2.85

Substrate

0.05

0.05

0.05

0.05


2. Mix by inversion and equilibrate using a suitably thermostatted spectrophotometer at 37 °C. Then add (in mL):

Solution

Blank

Test 1

Test 2

Test 3

Ultrapure water

0.10

Test

0.10

0.10

0.10

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.

Results

Calculations

Where:

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

Materials

     

References

  1. Drapeau, G.R., Methods in Enzymology, 45, 469 (1976). Trizma is a registered trademark of Sigma-Aldrich Co. LLC.) 03/17-1

 

Related Links