Enzymatic Assay of Trypsin Inhibitor

1. Objective

To standardize a procedure for the enzymatic assay for Trypsin Inhibitor.

2. Scope

This procedure applies to all products that have a specification for Trypsin Inhibition at Sigma-Aldrich, St. Louis.

3. Definitions

3.1. Purified Water - Water from a deionizing system, resistivity ~18MΩcm @25°C

3.2 Unit Definition – One Trypsin BAEE unit will produce a ΔA253nm of 0.001 per minute with BAEE as substrate at pH 7.6 at 25ºC in a reaction volume of 3.2 ml.

4. Discussion

Trypsin Inhibitor will inhibit the following reaction:
N-Benzoyl-L-Arginine Ethyl Ester + H2O   Trypsin   >N-Benzoyl-L-Arginine + Ethanol

5. Responsibilities

It is the responsibility of all trained Analytical Services 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°C, pH = 7.6 , A253nm, Light path = 1 cm

7.2 METHOD:
Spectrophotometric Rate Determination

7.3 REAGENTS:

7.3.1 67 mM Sodium Phosphate Buffer, pH 7.6 at 25ºC (BUFFER)
Prepare an 8.0 mg/mL solution in purified water using Sodium Phosphate, Monobasic, Anhydrous, such as Sigma-Aldrich Product Number S0751 . Adjust to pH 7.6 at 25ºC with 1 M NaOH.

7.3.2 0.25 mM N-Benzoyl-L-Arginine Ethyl Ester Solution (BAEE)
Prepare an 86 µg/mL solution in Reagent 7.3.1 using N-Benzoyl-L-Arginine Ethyl Ester, Hydrochloride, such as Sigma Product Number B4500.

7.3.3 1 mM Hydrochloric Acid Solution (HCl)
Prepare a 0.1% (v/v) solution in purified water using 1 N Hydrochloric Acid.

7.3.4 Trypsin Enzyme Solution (TRYP)
Immediately before use, prepare a solution containing 1.0 mg Protein/ml of Trypsin, such as Sigma Product Number T8003 , in cold reagent 7.3.3.

7.3.5 Trypsin Inhibitor Solution (INHB)
Immediately before use, prepare a solution containing 1.0 mg/ml of Trypsin Inhibitor in cold reagent 7.3.1.

7.3.5.1 When assaying Trypsin Inhibitor, Ovoinhibitor, Sigma Product Number T1886 , the diluent used is 200 mM Sodium Phosphate, Monobasic, pH 7.6 at 25ºC.

7.3.5.2 When assaying Trypsin Inhibitor, Type II-S, Sigma Product Number T9128 , prepare a solution containing 0.60 mg/ml of Trypsin Inhibitor in cold 7.3.1.

7.3.5.3 When assaying Trypsin Inhibitor, Defined, Sigma-Aldrich Product Number T7659 (Irvine equivalent, CR1333), use solution neat.

7.4 TEST METHOD

Inhibition Reaction

7.4.1 Pipette (in milliliters) the following reagents into a suitable containers:

  Uninhibited Test 1 Test 2 Test 3 Test 4 Test 5
Reagent 7.3.3 (HCl) 9.50 9.40 9.35 9.30 9.25 9.20
Reagent 7.3.5 (INHIB) ----- 0.10 0.15 0.20 0.25 0.30
Reagent 7.3.4 (TRYP) 0.50 0.50 0.50 0.50 0.50 0.50

 

7.4.2 Mix by inversion and allow to stand at 25ºC for a minimum of five minutes and no longer than six minutes.

7.4.3 Trypsin Inhibitor (Reagent 7.3.5) aliquots may be adjusted as necessary to achieve acceptable linearity.

Enzymatic Reaction:

7.4.4 Pipette (in milliliters) the following reagents into suitable cuvettes:

  Uninhibited Test 1 Test 2 Test 3 Test 4 Test 5 Blank
Reagent 7.3.2 (BAEE) 3.00 3.00 3.00 3.00 3.00 3.00 3.00
Reagent 7.3.3 (HCl) 0.10 0.10 0.10 0.10 0.10 0.10 0.20

 

7.4.5 Mix by inversion and equilibrate to 25ºC. Monitor the A253nm until constant, using a suitably thermostatted spectrophotometer. Then add:

  Uninhibited Test 1 Test 2 Test 3 Test 4 Test 5 Blank
7.4.1
(Uninhibited)
0.10 ----- ----- ----- ----- ----- -----
7.4.1 (Test 1) ----- 0.10 ----- ----- ----- ----- -----
7.4.1 (Test 2) ----- ----- 0.10 ----- ----- ----- -----
7.4.1 (Test 3) ----- ----- ----- 0.10 ----- ----- -----
7.4.1 (Test 4) ----- ----- ----- ----- 0.10 ----- -----
7.4.1 (Test 5) ----- ----- ----- ----- ----- 0.10 -----

 

7.4.6 Immediately mix by inversion and record the increase in A253nm for approximately 5 minutes. Obtain the ΔA253nm/minute using the maximum linear rate for the Uninhibited Solution using a minimum of four data points over a one minute time interval.

7.4.6.1 The uninhibited Trypsin activity should be within 85% of the release value for activity.

7.4.6.2 The maximum linear rate (corrected ?Abs253nm / minute) for the uninhibited solution should be between 0.0545 and 0.0835.

7.4.7 Apply time interval for the uninhibited solution determined in 7.4.6 to each of the tests and blank. Use these rates in calculation of inhibition. The Tests should result in 20% to 80% inhibition.

7.5 CALCULATIONS

7.5.1 BAEE Units/ml enzyme = (ΔA253nm/min (Test) - ΔA253nm/min (Blank))(10.0)(df)
(0.001)(0.10)(0.50)

 

7.5.2 BAEE Units/mg solid = BAEE Units/mL enzyme
mg solid/mL enzyme

 

7.5.3 Where:
df = Dilution factor
0.001 = The change in A253nm/minute per unit of Trypsin at pH 7.6 at 25ºC in a 3.2 mL reaction mix
0.10 = Volume (in milliliters) of Enzymatic Reaction Mixture used in step 7.4.5
10.0 = Total volume (in milliliters) of Inhibition Reaction in step 7.4.1
0.50 = Volume (in milliliters) of Trypsin used in 7.4.1

7.5.4 Mg Trypsin inhibitor/RM= (conc of Trypsin Inhibitor in mg/mL)(v)
10.0

 

7.5.5 Where:
RM = Reaction Mixture
conc = Concentration
v = Volume (in milliliters) of Trypsin Inhibitor solution used in step 7.4.1
10.0 = Total volume (in milliliters) of Inhibition Reaction in step 7.4.1

7.5.6 Plot the Trypsin activity (in BAEE units/mg Trypsin) vs. mg of Trypsin Inhibitor/RM. Record the y-intercept, slope and linear regression (R-square). The R-Squared value should be > or = to 0.95.

 

7.5.7 Mg Trypsin inhibitor/ml Inhibitor RM at 100% Inhibition= y-intercept
slope

 

7.5.8 Mg Trypsin inhibitor/mg s Inhibitor= mg Trypsin /ml Inhibitor RM
mg Trypsin Inhibitor/ml Inhibitor RM at 100% Inhibition

 

7.6. FINAL ASSAY CONTENTRATION:
In a 3.20 mL reaction mix, the final concentrations are 63 mM Sodium Phosphate, 0.23 mM BAEE, 0.002 mM HCl, 0.005 mg Trypsin, and 0.001 mg Trypsin Inhibitor.

8. References & Attachments

NA

9. Approval

Review, approvals and signatures for this document will be generated electronically using the EDMS. Print a “For Use” copy if hardcopy with signature verification is required.

Materials

     
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