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).
1.0 M Potassium phosphate monobasic solution (Catalog Number P8709)
1.0 M Potassium phosphate dibasic solution (Catalog Number P8584)
Potassium hydroxide (Catalog Number P1767)
Hydrochloric acid (Catalog Number H1758)
Casein (Catalog Number C7078)
L-Cysteine hydrochloride, monohydrate (Catalog Number C7880)
0.5 M Ethylenediaminetetraacetic acid solution, pH 8.0 (Catalog Number 03690)
Sodium Bicarbonate (Catalog Number S8875)
Potassium Phosphate, monobasic (Catalog Number P5379)
Trichloroacetic Acid solution [6.1 N, ~100% (w/v)], (Catalog Number T0699)
0.45 µM PVDF syringes
Cuvettes and thermostatted spectrophotometer
Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.
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:
Casein Solution [2.0% (w/v)] – Prepare a 20 mg/mL mixture of Casein (Catalog Number 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:
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 (Catalog Number 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)], (Catalog Number 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.
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.
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