Enzymatic Assay of Collagenase Using N-(3-[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGPA) as the Substrate (EC 22.214.171.124)
3.1. Purified Water = water from a deionizing system, resistivity > or = 18MΩ•cm @ 25ºC
3.2. Unit Definition = One unit hydrolyzes 1.0 μmole of furylacryloyl-Leu-Gly-Pro-Ala per minute at 25ºC at pH 7.5 in the presence of calcium ions.
3.3. FALGPA = N-(3-[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala
3.4. FAL = N-(3[2-Furyl]acryloyl)-Leu
7.1. CONDITIONS: pH = 7.5, T = 25°C, A345nm, Path Length = 1 cm
7.2. METHOD: Continuous Spectrophotometric Rate Determination
7.3.1. 50 mM Tricine with 10 mM Calcium Chloride and 400 mM Sodium Chloride, pH 7.5 at 25°C (Buffer)
Prepare an 8.96 mg/ml solution using Tricine, Sigma-Aldrich product number T0377, in purified water. Then add at 23.4 mg/ml Sodium Chloride, Sigma-Aldrich product number S9888, and adjust to pH 7.5 at 25°C with 1 M Sodium Hydroxide, Sigma-Aldrich product number S2567. Then add at 1.47 mg/ml Calcium Chloride, Sigma-Aldrich product number C3881 and recheck the pH of the solution at 25°C. If necessary, readjust to pH 7.5 at 25°C with 1 M Sodium Hydroxide, Sigma-Aldrich product number S2567 or 1 M Hydrochloric Acid, Sigma-Aldrich product number H3162.
7.3.2. 1.0 mM N-(3-[2Furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGPA)
Prepare a 1.0 mM solution using FALGPA, Sigma-Aldrich product number F5135, in Reagent 7.3.1, corrected for the water content of the specific lot. Complete dissolution will take at least 30 minutes with stirring. If necessary, readjust to pH 7.5 at 25°C with 1 M Sodium Hydroxide, Sigma-Aldrich product number S2567 or 1 M Hydrochloric Acid, Sigma-Aldrich product number H3162.
7.3.3. Enzyme Test Solution (Enzyme)
Immediately before use, prepare a solution containing 2 units/ml of Collagenase in cold purified water.
7.4.1. Pipette the following (in milliliters) into suitable containers:
|FALGPA (Reagent 7.3.2)||2.90||2.90|
Mix by inversion and equilibrate to 25°C. Monitor the A345nm until constant, using a suitably thermostatted spectrophotometer. Then add:
|Enzyme (Reagent 7.3.3)||0.10||------|
Immediately mix by inversion and record the decrease in A345nm for approximately 5 minutes. Obtain the maximum linear rate (Δ A345nm/minute) for both the test and blank using at least a one-minute interval and a minimum of 4 data points.
|7.5.1.||Units ml/Enzyme =||(ΔA345nm/min Test - ΔA345nm/min Blank) X (3.0) X (df)|
|(0.53) X (0.10)|
3.00 = Total volume (in milliliters) of the reaction mixture
df = Dilution factor of test
0.53 = Millimolar extinction coefficient of FALGPA at 345nm as determined experimentally by Sigma-Aldrich. It is directly related to the maximum decrease in absorabance per millimole of FALGPA at 345nm
0.10 = Volume (in milliliters) of enzyme used
|7.5.2.||Units/mg solid =||units/mL enzyme|
|mg solid/ml enzyme|
|Units/mg protein =||units/mL protein|
|mg protein/ml enzyme|
7.6. FINAL ASSAY CONCENTRATION
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.