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:

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)

V_t = Volume (in milliliters) of Ficin Solution (step 3) added

1.0 = Change in A₂₈₀ per unit as defined in the Unit Definition

2.

References

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