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

Analytical services personnel should 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 (Product No. 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 86 µg/mL in Reagent 7.3.1 using N-benzoyl-L-arginine ethyl ester, hydrochloride (Product No. B4500).

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

7.3.4 Trypsin Enzyme Solution (TRYP)
Immediately before use, prepare a solution containing 1 mg protein/mL of trypsin (Product No. T8003) in cold reagent 7.3.3.

7.3.5 Trypsin Inhibitor Solution (INHB)
Immediately before use, prepare a solution containing 1 mg/mL of trypsin Inhibitor in cold reagent 7.3.1.

7.3.5.1 When assaying trypsin inhibitor, ovoinhibitor (Product No. T1886), the diluent is 200 mM sodium phosphate, monobasic, pH 7.6 at 25 ºC.

7.3.5.2 When assaying trypsin inhibitor, Type II-S (Product No. 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 No. T7659 (Irvine equivalent, CR1333), use the 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 incubate 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 absorbance 253 nm/minute) for the uninhibited solution should be from 0.0545 to 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 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, the final concentrations are as follows: 63 mM sodium phosphate, 0.23 mM BAEE, 0.002 mM HCl, 0.005 mg trypsin, and 0.001 mg trypsin Inhibitor.