Enzymatic Assay of Ficin (EC 3.4.22.3)
Description
This procedure may be used for all ficin products.
The spectrophotometric stop rate detemination (A280, Light path = 1 cm) is based on the following reaction:
Unit Definition = One unit of ficin will produce a ΔA280 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 mL (1 cm light path).
Reagents and Equipment Required
1.0 M Potassium phosphate monobasic solution (Product No. P8709)
1.0 M Potassium phosphate dibasic solution (Product No. P8584)
Potassium hydroxide (Product No. P1767)
Hydrochloric acid (Product No. H1758)
Casein (Product No. C7078)
L-Cysteine hydrochloride, monohydrate (Product No. C7880)
0.5 M Ethylenediaminetetraacetic acid solution, pH 8.0 (Product No. 03690)
Sodium Bicarbonate (Product No. S8875)
Potassium Phosphate, monobasic (Product No. P5379)
Trichloroacetic Acid solution [6.1 N, ~100% (w/v)] (Product No. T0699)
0.45 µM PVDF syringes
Cuvettes and thermostatted spectrophotometer
Precautions
Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.
Preparation Instructions
(Storage/Stability)
Use ultrapure water (≥18 MΩ×cm resistivity at 25 °C) for the preparation of reagents.
Phosphate Buffer (100 mM Potassium Phosphate Buffer, pH 7.0 at 37 °C)
To prepare 100 mL:
- Add 3.85 mL of 1.0 M Potassium phosphate monobasic solution (Product No. P8709).
- Add 6.15 mL of 1.0 M Potassium phosphate dibasic solution (Product No. P8584).
- Add ultrapure water to make up the final volume to 100 mL.
- Adjust the pH to 7.0 at 37 °C using 1 M KOH or HCl.
Casein Solution [2.0% (w/v)] – Prepare a 20 mg/mL mixture of Casein (Product No. C7078) in Phosphate Buffer. Heat gently with stirring to 80–85 °C for ~10 minutes until a homogenous dispersion is achieved. DO NOT BOIL. Adjust volume back to original volume with ultrapure water.
L-Cys/EDTA Solution (250 mM L-Cysteine and 250 mM EDTA)
To prepare 25 mL:
- Add 1.10 g of L-Cysteine hydrochloride, monohydrate (Product No. C7880).
- Add 12.5 mL of 0.5 M Ethylenediaminetetraacetic acid solution, pH 8.0 (Product No. 03690).
- Add 10 mL of ultrapure water.
- pH to 7.0 at 37 °C with solid Sodium Bicarbonate (Product No. S8875) and/or 0.1 M HCl.
Enzyme Diluent (1 M Potassium Phosphate Buffer, pH 7.0 at 37 °C) – Prepare a 136 mg/mL solution in ultrapure water of Potassium Phosphate, monobasic (Product No. P5379). Adjust pH to 7.0 at 37 °C with 5 M KOH.
TCA Solution [5.0% (w/v) Trichloroacetic Acid Solution] – A 1:20 dilution of Trichloroacetic Acid solution [6.1 N, ~100% (w/v)] (Product No. T0699) with ultrapure water.
Ficin Solution – Immediately before use, prepare a solution containing 0.04–0.08 unit/mL of Ficin in cold Enzyme Diluent.
Procedure
Final Assay Concentrations – 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 unit/mL of ficin.
1. Prepare a Reaction Cocktail by pipetting the following reagents into a suitable container:
Adjust to pH 7.0 at 37 °C with 1 M HCl or KOH. Volume of the reaction cocktail may be adjusted, if necessary.
2. Pipette the following reagents into suitable containers:
3. Mix by swirling and equilibrate to 37 °C in a suitably thermostatted water bath.
4. Then add:
5. Immediately mix by swirling and incubate at 37 °C in a suitably thermostatted water bath for exactly 20 minutes.
6. Then add:
7. Mix by swirling and incubate at 37 °C in a suitably thermostatted water bath for 30 minutes.
8. Filter all the Tests and Blank through a 0.45 µM PVDF syringe filter into suitable cuvettes.
9. Record the A280 of all the Tests and Blank using a suitable spectrophotometer blanked against air.
Results
Calculations
1.
Where:
df = Dilution factor of enzyme
20 = Time of assay (in minutes)
Vt = Volume (in milliliters) of Ficin Solution (step 3) added
1.0 = Change in A280 per unit as defined in the Unit Definition
2.
References
Per continuare a leggere, autenticati o crea un account.
Non hai un Account?