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 @ 25ºC

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 = 25ºC, 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, Sigma-Aldrich Product Number such as 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, Sigma-Aldrich Product Number such as T1503, in purified water. Adjust pH to 8.4 at 25ºC with 5N HCl.

7.3.3   0.2 mg/ml Trypsin (Trypsin) 
Immediately before use, prepare a 0.2 mg/ml solution of Trypsin, Sigma-Aldrich Product Number such as 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, Sigma-Aldrich Product Number such as H3162, in purified water.

7.3.5   0.001 N HCl (HCl-2) 
Prepare a 1:1000 dilution of 1 N HCl, Sigma-Aldrich Product Number such as 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, Sigma-Aldrich Product Number such as S0751, in purified water. Adjust pH to 7.6 at 25ºC with 1N NaOH.

7.3.7   0.248 mM BAEE (BAEE) 
Prepare a 0.085 mg/ml solution of BAEE, Sigma-Aldrich Product Number such as 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 0-5oC 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 0-5ºC for 24 – 28 hours

7.4.3.2.    After incubation, dilute 0.10 ml of Reagent 7.3.8. (Trypsinogen) to 1.0ml 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 25ºC 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.

Materials

     
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