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 ΔA253nm 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 , 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 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:
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:
7.4.5 Mix by inversion and equilibrate to 25 ºC. Monitor the A253nM until constant, using a suitably thermostatted spectrophotometer. Then add:
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 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
7.5.3 Where:
df = Dilution factor
0.001 = The change in A253nM/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
7.5.5 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.
7.6. 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.
若要繼續閱讀，請登入或建立帳戶。
還沒有帳戶？