To standardize a procedure for the determination of the enzymatic assay of trypsinogen.
This scope of this procedure applies to products that have a specification for the enzymatic activity of trypsinogen.
3.1 BAEE = Nα-Benzoyl-L-Arginine Ethyl Ester
3.2 Purified water = water from a deionizing system, resistivity ~18MΩ•cm @ 25 ºC
NA
It is the responsibility of all trained Analytical Services laboratory personnel to follow this protocol as written.
Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions.
7.1 CONDITIONS: T = 25 ºC, pH = 7.6, A253nm, Light path = 1 cm
7.2.METHOD: Continuous Spectrophotometric Rate Determination
7.3. REAGENTS
7.3.1 1 M Calcium Chloride (CaCl2)
Prepare a 147 mg /mL solution of Calcium Chloride (Product No. C3881) in purified water.
7.3.2 400 mM Trizma Buffer, pH 8.4 at 25 ºC (Activation Buffer)
Prepare a 48.5 mg/mL solution of Trizma (Product No. T1503) in purified water. Adjust pH to 8.4 at 25 ºC with 5N HCl.
7.3.3 0.2 mg/mL Trypsin (Trypsin)
Immediately before use, prepare a 0.2 mg/mL solution of Trypsin (Product No. T9201) in Reagent 7.3.4 (HCl-1).
7.3.4 0.005 N HCl (HCl-1)
Prepare a 1:200 dilution of 1 N HCl (Product No. H3162) in purified water.
7.3.5 0.001 N HCl (HCl-2)
Prepare a 1:1000 dilution of 1 N HCl (Product No. H3162) in purified water.
7.3.6 67 mM Sodium Phosphate, pH 7.6 at 25 ºC (Buffer)
Prepare a 8.4 mg/mL solution of Sodium Phosphate, Monobasic (Product No. B4500) in Reagent 7.3.6 (Buffer).
7.3.8 5 mg Protein/mL Trypsinogen (Trypsinogen)
Immediately before use, prepare a 5 mg Protein/mL solution of Trypsinogen in Reagent 7.3.4 (HCl-1).
7.4 TEST METHOD
7.4.1. Prepare Activating Mixture as follows with stirring (in milliliters):
Reagent 7.3.1. (CaCl2) | 2.0 |
Reagent 7.3.2. (Activation Buffer) | 38.0 |
Reagent 7.3.3. (Trypsin) | 2.0 |
7.4.2. TOTAL TRYPSIN ACTIVITY:
7.4.2.1. Add 0.1 mL of Reagent 7.3.8. (Trypsinogen) to 1.0 mL of Activating Mixture from step 7.4.1. Incubate at 0-5 °C for 24–28 hours.
7.4.2.2. After incubation, dilute 0.2 mL to 10.2 mL with Reagent 7.3.5 (HCl-2) and measure trypsin activity as detailed in step 7.4.4.
7.4.3. FREE TRYPSIN ACTIVITY:
7.4.3.1. Let Activating Mixture from step 7.4.1 and Reagent 7.3.8. (Trypsinogen) solutions incubate separately at 0-5 ºC for 24–28 hours
7.4.3.2. After incubation, dilute 0.10 mL of Reagent 7.3.8. (Trypsinogen) to 1.0 mL with Reagent 7.3.5 (HCl-2) and measure trypsin activity as detailed in step 7.4.4.
7.4.4. Pipette (in milliliters) the following reagents into quartz cuvettes:
Test | Blank | |
Reagent 7.3.7 (BAEE) | 3.00 | 3.00 |
Reagent 7.3.5 (HCl-2) | ---- | 0.20 |
7.4.5. Mix by inversion and incubate at 25 ºC using a suitable thermostatted spectrophotometer. Then add:
Enzyme Solution from 7.4.2.2 or 7.4.3.2 | 0.20 | ---- |
Immediately mix by inversion and record the increase in A253nm for approximately 10 minutes. Obtain the ΔA253nm / minute using the maximum linear rate for both the Test and the Blank.
7.5 CALCULATIONS
7.5.1. | Units/mL enzyme= | (ΔA253nm/min Test - ΔA253nm/min Blank)(df) (0.001)(0.2) |
where,
df = Dilution Factor
0.001 = Micromolar extinction coefficient for BAEE at 253nm
0.2 = Volume (in milliliters) of enzyme used
7.5.2. | Units/mg solid = | Units/mL enzyme mg solid/mL enzyme |
7.5.3. | Units/mg protein = | Units/mg Solid mg protein/mg Solid |
7.6 UNIT DEFINITION
One BAEE unit is equal to a ΔA253 of 0.001 per min with BAEE as substrate at pH 7.6 at 25 °C and a reaction volume of 3.2 mL (1 cm light path).
Replaces SPBAEE15.
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