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 our products having a specification for Trypsin Inhibition.

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 ΔA_253nm of 0.001 per minute with BAEE as substrate at pH 7.6 at 25 ºC 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 , A_253nM, 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 25 ºC (BUFFER)
Prepare an 8.0 Mg/mL solution in purified water using Sodium Phosphate, Monobasic, Anhydrous, such as Product Number S0751 . Adjust to pH 7.6 at 25 ºC 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 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 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, Product Number T1886 , the diluent used is 200 mM Sodium Phosphate, Monobasic, pH 7.6 at 25 ºC.

7.3.5.2 When assaying Trypsin Inhibitor, Type II-S, 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, 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 25 ºC 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 25 ºC. Monitor the A_253nM 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 A_253nM for approximately 5 minutes. Obtain the ΔA_253nM/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 A_253nM/minute per unit of Trypsin at pH 7.6 at 25 ºC 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.