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HomeEnzyme Activity AssaysEnzymatic Assay of Ficin (EC 3.4.22.3)

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:

ficin-eq

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:

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.

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.

ficin-units

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.

ficin-solid
Materials
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References

1.
Liener IE, Friedenson B. 1970. [17] Ficin.261-273. https://doi.org/10.1016/0076-6879(70)19020-3
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