HomeEnzyme Activity AssaysEnzymatic Assay of Ficin (EC

Enzymatic Assay of Ficin (EC


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


Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Preparation Instructions


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 (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:

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.


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




Liener IE, Friedenson B. 1970. [17] Ficin.261-273.
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