Enzymatic Assay of Collagenase (EC 3.4.24.3) using FALGPA (N-(3-[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala)

This procedure may be used for Collagenase products.

The continuous spectrophotometric rate determination (A₃₄₅, Light path = 1 cm) is based where:

FALGPA = N-(3-[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala
FAL = N-(3[2-Furyl]acryloyl)-Leu

Unit Definition – One unit of Collagenase hydrolyzes 1.0 µmole of FALGPA per minute at 25 °C at pH 7.5 in the presence of calcium ions.

Reagents and Equipment Required

• Tricine (Catalog Number T0377)
• 5.0 M Sodium chloride solution (Catalog Number S6546)
• 1 M Sodium hydroxide solution (Catalog Number S2567)
• 1 M Calcium chloride solution (Catalog Number 21115)
• 1 M Hydrochloric acid solution (Catalog Number H3162)
• FALGPA (Catalog Number F5135)

Precautions

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

Preparation Instructions

Use ultrapure water (≥18 MΩxcm resistivity at 25 °C) for the preparation of reagents.

Buffer (50 mM Tricine with 10 mM Calcium Chloride and 400 mM Sodium Chloride, pH 7.5 at 25 °C) – Weigh ~2.24 g of Tricine (Catalog Number T0377) in a suitable beaker. Add 200 mL of ultrapure water. Add 20 mL of 5.0 M Sodium chloride solution (Catalog Number S6546). Mix well and adjust the solution to pH 7.5 at 25 °C using 1–5 M NaOH or HCl. After pH adjustment, add 25 mL of 100 mM Calcium chloride solution, prepared using Catalog Number 21115. Add purified water to a final volume of 250 mL. Adjust to pH 7.5 at 25 °C using 1–5 M NaOH or HCl, if necessary.

FALGPA solution (1.0 mM N-(3-[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala) – Prepare a corrected 0.48 mg/mL (1.0 mM) solution using N-(3-[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala (Catalog Number F5135) in Buffer. Correct the concentration based on the water content of the specific lot of FALGPA used. Stir for at least 30 minutes for complete dissolution. Adjust to pH 7.5 at 25 °C with 1 M Sodium hydroxide solution (Catalog Number S2567) or 1 M Hydrochloric acid solution (Catalog Number H3162), if necessary.

Enzyme Solution – Immediately before use, prepare a solution containing 2 units/ml of Collagenase in cold (2–8 °C) ultrapure water.

Procedure

In a 3.00 mL reaction mix, the final concentrations are 48.3 mM tricine, 9.67 mM calcium chloride, 387 mM sodium chloride, 0.967 mM FALGPA, and 0.20 unit collagenase.

1. Pipette the following into suitable containers:

Reagent	Blank (mL)	Test 1 (mL)	Test 2 (mL)	Test 3 (mL)
FALGPA solution	2.90	2.90	2.90	2.90
ultrapure water	0.10	–	–	–

2. Mix by inversion and equilibrate to 25 °C using a suitably thermostatted spectrophotometer.

3. Then add:

Reagent	Blank (mL)	Test 1 (mL)	Test 2 (mL)	Test 3 (mL)
Enzyme solution	–	0.10	0.10	0.10

4. Immediately mix by inversion and record the decrease in A₃₄₅ for ~5 minutes. Obtain the maximum linear rate (ΔA₃₄₅/minute) for all the Tests and the Blank using at least a one-minute interval and a minimum of 4 data points.

Results

Calculations

1.

Units/mL enzyme =		(ΔA345/min Test – ΔA345/min Blank) (3.00) (df)
		(0.53) (0.10)

where:
3.00 = Total volume (mL) of the reaction mixture
df = Dilution factor
0.53 = Millimolar extinction coefficient of FALGPA at 345 nm determined experimentally. It is directly related to the maximum decrease in absorbance per millimole of FALGPA at 345 nm.
0.10 = Volume (mL) of Enzyme solution used

2.

Units/mg solid =		units/mL enzyme
		mg solid/mL enzyme

1. Van Wart HE, Steinbrink D. 1981. A continuous spectrophotometric assay for Clostridium histolyticum collagenase. Analytical Biochemistry. 113(2):356-365. https://doi.org/10.1016/0003-2697(81)90089-0