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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 (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

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 (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.

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:
 

Reagent Volume (ml)
Casein Solution 40.0
L-Cys/EDTA Solution 4.00
Enzyme Diluent 12.0
Ultrapure Water 16.0


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:
 

Reagent Volume (ml)
Test 1 Test 2 Test 3 Blank
Reaction Cocktail 3.60 3.60 3.60 3.60
Enzyme Diluent 0.10 0.05 0.40


3. Mix by swirling and equilibrate to 37 °C in a suitably thermostatted water bath.

4. Then add:
 

Reagent Volume (ml)
Test 1 Test 2 Test 3 Blank
Ficin Solution 0.30 0.35 0.40


5. Immediately mix by swirling and incubate at 37 °C in a suitably thermostatted water bath for exactly 20 minutes.  

6. Then add:
 

Reagent Volume (ml)
Test 1 Test 2 Test 3 Blank
TCA Solution 6.00 6.00 6.00 6.00


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.

Materials

     

 References

  1. Liener, I.E., and Friedenson, B., Methods in Enzymology, XIX, 261-273 (1970).

 

08/17-1

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