Enzymatic Assay of Protease Using Casein As a Substrate
3.1 UNIT DEFINITION - One unit will hydrolyze casein to produce color equivalent to 1.0 µmole of tyrosine per minute at pH 7.5 at 37ºC. Color per Folin & Ciocalteau’s reagent.
3.2 Purified Water - purified water from a deionizing system with a resistivity >18 MΩּcm-1, or equivalent.
7.1 CONDITIONS: T = 37ºC, pH=7.5, A660nm Light Path = 1cm
7.2 METHOD: Spectrophotometric Stop Rate Assay
7.3.1 50 mM Potassium Phosphate buffer, pH 7.5 at 37ºC (Buffer)
Prepare 11.4 mg/mL in purified water using Potassium Phosphate, Dibasic, Trihydrate, Sigma-Aldrich Product Number P5504. Adjust to pH 7.5 at 37ºC with 1 N HCl.
7.3.2 0.65% (w/v) Casein Solution (Casein)
22.214.171.124 Prepare 6.5 mg/mL in reagent 7.3.1 (Buffer) using Casein, Sigma-Aldrich Product Number C7078.
126.96.36.199 Heat gently with stirring to 80-85ºC for approximately 10 minutes until a homogeneous dispersion is achieved. Do not boil.
188.8.131.52 Adjust the pH to 7.5 at 37ºC, if necessary, with 0.1 N NaOH or 0.1 N HCl.
7.3.3 110 mM Trichloroacetic Acid Reagent (TCA)
Prepare 1:55 dilution of Trichloroacetic Acid, 6.1N, approximately 100% (w/v), Sigma-Aldrich Product Number T0699, with purified water.
7.3.4 0.5 M Folin & Ciocalteu’s Phenol Reagent (F-C)
Prepare a 1:4 dilution of 2 M Folin & Ciocalteu’s Phenol Reagent, Sigma-Aldrich Product Number F9252, with purified water.
7.3.5 500 mM Sodium Carbonate Solution (Na2CO3)
Prepare 53 mg/mL in purified water using Sodium Carbonate, Anhydrous, Sigma-Aldrich Product Number S2127.
7.3.6 10 mM Sodium Acetate Buffer with 5 mM Calcium Acetate, pH 7.5 at 37ºC (Enzyme Diluent)
Prepare 1.4 mg/mL Sodium Acetate, Trihydrate, Sigma-Aldrich Product Number S8625, and 0.8 mg/mL Calcium Acetate, Sigma-Aldrich Product Number C1000, in purified water. Adjust the pH to 7.5 at 37ºC with 0.1 M Acetic Acid or 0.1 N NaOH.
7.3.7 1.1 mM L-Tyrosine Standard (Std Soln)
Prepare 0.2 mg/mL L-Tyrosine, Free Base, Sigma-Aldrich Product Number T3754, in purified water. Heat gently (do not boil) until tyrosine dissolves. Cool to room temperature.
7.3.8 Protease Enzyme Solution
Immediately before use, prepare a solution containing 0.1 – 0.2 units/mL of Protease in cold Reagent 7.3.6 (Enzyme Diluent). For samples where little or no protease detection is expected, prepare sample at 10 mg solid/mL in cold Reagent 7.3.6 (Enzyme Diluent).
7.4 ASSAY PROCEDURE
7.4.1 Pipette the following into suitable vials (in milliliters):
|Casein (Reagent 7.3.2)||5.00||5.00||5.00||5.00|
7.4.2 Let the vials equilibrate in a suitably thermostated water bath at 37ºC for about 5 minutes, then add:
|Enzyme Solution (Reagent 7.3.8)||1.00||0.70||0.50||-----|
7.4.3 Mix by swirling and incubate at 37ºC for exactly 10 minutes. Then add:
|TCA (Reagent 7.4.3)||5.00||5.00||5.00||5.00|
|Enzyme Solution (Reagent 7.3.8)||-----||0.30||0.50||1.00|
7.4.4 Mix by swirling and incubate at 37ºC for about 30 minutes.
7.4.5 Filter each solution using a 0.45 μm syringe filter and use the filtrate in Step 7.4.6.
7.4.6 Pipette the following reagent into 4 dram vials (in milliliters): For more consistent results, add F-C (Reagent 7.3.4) immediately following the addition of Na2CO3 (Reagent 7.3.5).
|Test Filtrate (Step 7.4.5)||2.00||2.00||2.00||-----|
|Blank Filtrate (Step 7.4.5)||-----||-----||-----||2.00|
|Na2CO3 (Reagent 7.3.5)||5.00||5.00||5.00||5.00|
|F-C (Reagent 7.3.4)||1.00||1.00||1.00||1.00|
7.4.7 Prepare a standard curve by pipetting the following reagents into suitable vials (in milliliters).
184.108.40.206 For impurity samples, low standards may be added as needed:
220.127.116.11 For more consistent results, add F-C (Reagent 7.3.4) immediately following the addition of Na2CO3 (Reagent 7.3.5).
|Std 1||Std 2||Std 3||Std 4||Std 5||Std Blank|
|Std Soln (Reagent 7.3.7)||0.05||0.10||0.20||0.40||0.50||0.00|
|Na2CO3 (Reagent 7.3.5)||5.00||5.00||5.00||5.00||5.00||5.00|
|F-C (Reagent 7.3.4)||1.00||1.00||1.00||1.00||1.00||1.00|
7.4.8 Mix by swirling and incubate Blanks, Standards, and Tests at 37ºC for 30 minutes. Remove the vials and allow to cool to room temperature.
7.4.9 Filter each Blank, Standard, and Test using a 0.45 μm syringe filter into suitable cuvettes.
7.4.10 Record the A660nm of each Test, Standard, and Blank solution.
7.5.1 Standard Curve
|18.104.22.168.||ΔA660nm(Standard)=||ΔA660nm(Standard) - ΔA660nm(Standard Blank)|
|22.214.171.124.||Plot the ΔA660nm(Standard) vs μmoles of Tyrosine|
7.5.2 Sample Determination
|126.96.36.199.||ΔA660nm(Test)=||ΔA660nm(Test) - ΔA660nm(Test Blank)|
|188.8.131.52.||Units/mL enzyme =||(μmole Tyrosine equivalents released)(11)|
11 = Total volume of assay in milliliters
2 = Volume (in milliliters) used in Colorimetric Determination
1 = volume of enzyme used for assay
10 = time (in minutes) of assay
|184.108.40.206.||Units/mg solid =||Units/mL enzyme|
|mg solid/ml enzyme|
|220.127.116.11.||Units/mg protein =||Units/mL enzyme|
|mg protein/ml enzyme|
7.6 FINAL ASSAY CONCENTRATIONS
In a 6.00 ml reaction mix, the final concentrations are 42 mM potassium phosphate, 0.54% (w/v) casein, 1.7 mM sodium acetate, 0.8 mM calcium acetate, and 0.1 –0.2 unit protease.
8.1 Anson, M.L., (1938) J. Gen. Physiol. 22, 79-89
8.2 Folin, O. and Ciocalteau, V., (1929) J. Biol. Chem. 73, 627