Enzymatic Assay of Ficin
1. OBJECTIVE
To standardize a procedure for determining the activity of Ficin by enzymatic analysis.
2. SCOPE
This procedure applies to all products that have a specification for Ficin activity.
3.1 Purified Water = water from a deionizing system, resistivity > or = 18MΩ•cm @ 25ºC
4. DISCUSSION
Casein + H2O Ficin > Amino Acids
5. RESPONSIBILITIES
It is the responsibility of trained Analytical Services laboratory personnel to follow this procedure as written.
6. SAFETY
Refer to Material Safety Data Sheets (MSDS) for hazards and appropriate handling precautions.
7.1 CONDITIONS:
T = 37°C, pH = 7.0, A280nm, Light path = 1 cm
7.2 METHOD:
Spectrophotometric Stop Rate Determination
7.3 REAGENTS:
7.3.1 100mM Potassium Phosphate Buffer, pH 7.0 at 37°C (Buffer)
Prepare a 13.6 mg/ml solution in purified water of Monobasic Potassium Phosphate, using a Sigma-Aldrich Product Number such as P5379. Adjust to pH 7.0 at 37°C with 5N KOH.
7.3.2 2.0% (w/v) Casein Solution (Casein)
Prepare a 20 mg/ml in Reagent 7.3.1. (Buffer) of Casein, using a Sigma-Aldrich Product Number such as C7078. Heat gently with stirring to 80-85°C for approximately 10 minutes until a homogenous dispersion is achieved. DO NOT BOIL. Adjust volume back to original volume with purified water.
7.3.3 250mM L-Cysteine and 250mM EDTA Solution (L-Cys/EDTA)
Prepare a 44 mg/ml solution in purified water of Monohydrate L-Cysteine hydrochloride, using a Sigma-Aldrich Product Number such as C7880 and 104 mg/ml Ethylenediaminetetraacetic Acid, tetrasodium salt hydrate, Sigma-Aldrich Product Number such as ED4SS. Adjust pH to 7.0 at 37°C with solid Sodium Bicarbonate, Sigma-Aldrich Product Number such as S8875 and/or 0.1N HCl.
7.3.4 M Potassium Phosphate Buffer, pH 7.0 at 37°C (Enzyme Diluent)
Prepare a 136 mg/ml solution in purified water of Monobasic Potassium Phosphate, using a Sigma-Aldrich Product Number such as P5379. Adjust pH to 7.0 at 37°C with 5N KOH.
7.3.5 5.0% (w/v) Trichloroacetic Acid Solution (TCA)
A 1:20 dilution of Trichloroacetic Acid, 6.1N, approximately 100% (w/v), using a Sigma-Aldrich Product Number such as T0699, with purified water.
7.3.6 Ficin Sample/Control Solutions (Enzyme)
Immediately before use, prepare a solution containing 0.04 – 0.08 unit/ml of Ficin in cold Reagent 7.3.4 (Enzyme Diluent).
7.4 TEST METHOD
7.4.1 Pipette (in milliliters) the following reagents into a suitable container for a cocktail:
| Reagent 7.3.2. (Casein) | 40.0 |
| Reagent 7.3.3. (L-Cys/EDTA) | 4.00 |
| Reagent 7.3.4. (Enzyme Diluent) | 12.0 |
| Purified Water | 16.0 |
Adjust to pH 7.0 at 37°C with HCl or KOH.
7.4.2 Pipette (in milliliters) the following reagents into suitable containers:
| Test-1 | Test-2 | Test-3 | Blank | |
| Cocktail | 3.60 | 3.60 | 3.60 | 3.60 |
| Reagent 7.3.4. (Enzyme Diluent) | 0.10 | 0.05 | ------ | 0.40 |
7.4.3 Mix by swirling and equilibrate to 37°C. Then add (in milliliters):
| Reagent 7.3.6. (Enzyme) | 0.30 | 0.35 | 0.40 | ------ |
7.4.4 Immediately mix by swirling and incubate at 37°C for exactly 20 minutes. Then add:
| Reagent 7.3.5. (TCA) | 6.00 | 6.00 | 6.00 | 6.00 |
7.4.5 Mix by swirling and incubate at 37°C for 30 minutes. Filter through 0.45μm PVDF syringe filter into suitable cuvettes.
7.4.6 Record the A280nm of each Test and Blank solution.
7.5 CALCULATIONS
| 7.5.1 | Units/ml enzyme = | (A280nm Test - A280nm Blank)(df) |
| (20)(0.4)(1.0) |
where:
df = Dilution Factor of enzyme
20 = Time of assay (in minutes)
0.4 = Volume (in milliliters) of enzyme used
1.0 = Change in A280nm per unit as per the Unit Definition
| 7.5.2 | Units/mg solid = | Units/mL enzyme |
| mg solid/mL enzyme |
| 7.5.3 | Units/mg protein = | Units/mL enzyme |
| mg protein/mL enzyme |
7.6 FINAL ASSAY CONCENTRATION :
In a 4.00 ml reaction volume, the final concentrations are 1.0% (w/v) casein, 300 mM potassium phosphate, 12.5 mM L-cysteine, 12.5 mM EDTA, and 0.012-0.032 units/ml ficin.
7.7 Unit Definition = One unit will produce a ΔA280nm of 1.0 per minute at pH 7.0 at 37°C when measuring TCA soluble products from casein in a final volume of 10 mls. (1 cm light path).
8.1 Replaces SSCASE10
8.2 Liener, I.E. and Friedenson, B. (1970) Methods in Enzymology, XIX, 261-273.
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