Enzymatic Assay of Protease, Product No. P2922
1. OBJECTIVE
To standardize a procedure for the enzymatic assay of Protease activity with N-t-Boc-L-glutamic acid α-phenyl ester substrate.
2.1. This assay procedure is to be used to assay Endoproteinase Glu-C, Sigma-Aldrich Product Number P2922.
3.1. Purified Water - water from a deionizing system, resistivity ~18MΩ•cm @ 25ºC
3.2. Unit Definition – One unit will hydrolyze 1 μmole of N-t-Boc-L-glutamic acid α-phenyl ester per min at pH 7.8 at 37 °C. One unit is equivalent to ~0.004 casein digestion unit.
4. DISCUSSION
N -t -BOC -GAP + H2O Protease >N -t -BOC-L- Glutamic Acid + Phenol
Abbreviations:
BOC = N-tert-Butoxy-Carbonyl
N-t-BOC-GAPE = N-t-BOC-L-Glutamic Acid α-Phenyl Ester
5. RESPONSIBILITIES
It is the responsibility of all trained Analytical Services laboratory personnel to follow this protocol as written.
6. SAFETY
Refer to the Material Safety Data Sheet (MSDS) for hazards and appropriate handling precautions.
7.1 CONDITIONS:T=37ºC, pH=7.8, A270nm, Light Path=1 cm
7.2. METHOD: Continuous Spectrophotometric Rate Determination
7.3 REAGENTS
7.3.1 200 mM Tris-Phosphate Buffer, pH=7.8 at 37ºC (Buffer)
Prepare a 24.22 mg/mL solution of Trizma Base, using Sigma-Aldrich Product Number such as T1503, in purified water. Mix well and adjust to pH 7.8 at 37ºC with Reagent 7.3.2. (Acid)
7.3.2 5N Phosphoric Acid (Acid)
Prepare a 0.11 ml/ml solution of Phosphoric Acid, using Sigma-Aldrich Product Number such as 215104, in purified water.
7.3.3 60 mM N-t-BOC-L-Glutamic Acid α-Phenyl Ester Substrate Solution (Substrate)
Prepare 19.4 mg/mL solution of N-t-BOC-L-Glutamic Acid α-Phenyl Ester, using BACHEM Product Number such as A4540 in 1,4-Dioxane using Sigma-Aldrich Product Number such as 34857. PREPARE FRESH. **Caution: 1,4 Dioxane is a peroxide forming compound. Refer to SOP 1.09 for peroxide forming compounds.
7.3.4 Protease Enzyme Solution (Enzyme)
Immediately before use, prepare a solution containing 0.1-2.0 units/mL of Protease in purified water.
7.4. TEST METHOD
7.4.1. Pipette (in milliliters) into suitable cuvettes:
| Test | Blank | |
| Reagent 7.3.1 (Buffer) | 2.85 | 2.85 |
| Reagent 7.3.3 (Substrate) | 0.05 | 0.05 |
7.4.2. Mix by inversion and equilibrate to 37ºC. Then add (in milliliters):
| Test | Blank | |
| Reagent 7.3.4 (Enzyme) | 0.10 | ---- |
| Purified Water | ---- | 0.10 |
7.4.3. Immediately mix by inversion and record the decrease in A270nm for approximately 5 minutes. Obtain the A270nm /minute using the maximum linear rate for both the Test and Blank using a minimum of 4 data points over a one minute time interval.
7.5 CALCULATIONS
| 7.5.1 | Units/ml enzyme = | (ΔA270nm/min Test - ΔA270nm/min Blank)(3)(df) |
| (1.5)(0.1) |
where:
3 = Total Volume (in milliliters) of assay
df = Dilution factor
1.5 = Millimolar extinction coefficient of Phenol at 270nm
0.1 = Volume (in milliliters) of enzyme used
| 7.5.2 | Units/mg solid = | Units/mL enzyme |
| mg solid/mL enzyme |
| 7.5.3 | Units/mg protein = | Units/mg Solid |
| mg protein/mg Solid |
7.6 FINAL ASSAY CONTENTRATION
In a 3.00 ml reaction mix, the final concentrations are 190 mM Tris, 1 mM N-t-BOC-L-Glutamic acid α-phenyl ester, 1.7% 1,4-dioxane, and 0.01-0.2 units protease.
8.1. Drapeau, G. R. (1976) Methods in Enzymology, 45, 469.
8.2. Replaces SPGLUT04.
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