Enzymatic Assay of Trypsinogen
1. Objective
To standardize a procedure for the determination of the enzymatic assay of trypsinogen.
2. Scope
This scope of this procedure applies to products that have a specification for the enzymatic activity of trypsinogen.
3. Definitions
3.1 BAEE = Nα-Benzoyl-L-Arginine Ethyl Ester
3.2 Purified water = water from a deionizing system, resistivity ~18MΩ•cm @ 25 ºC
4. Discussion
NA
5. Responsibilities
It is the responsibility of all trained Analytical Services laboratory personnel to follow this protocol as written.
6. Safety
Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions.
7. Procedure
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).
8. References & Attachments
Replaces SPBAEE15.
9. Approval
Review, approvals and signatures for this document will be generated electronically using EDMS. Print a “For Use” copy if hardcopy with signature verification is required.