Trypsin Inhibitors
Cell Culture Application
Trypsin inhibitors are used in cell culture applications to further inhibit tryptic activity during cell dissociation to prevent cell damage/death.
Procedure:
After trypsinizing cells, resuspend cells in 1 mL trypsin inhibitor solution (1 mg/mL using either a balanced salt solution or serum free media) for every mL of trypsin solution used for dissociation. Centrifuge the cell suspension at 1000 rpm for 5 minutes. A cell pellet should form. Remove as much of the trypsin inhibitor as possible and resuspend the pellet in serum-free medium. Culture cells as desired.
Assay Method for Trypsin Inhibitor Activity
The activity of most trypsin inhibitor preparations is determined by a continuous rate spectrophotometric assay and expressed as the inhibition of BAEE units.
Unit Definition: One BAEE unit will produce a ΔA253 of 0.001 per min at pH 7.6 and 25 °C using BAEE as a substrate. Reaction volume = 3.2 mL.
Conditions
Temp = 25 °C, pH = 7.6, A= 253 nm, Light path = 1 cm
In a 3.2 mL reaction mix, the final concentrations are 63 mM sodium phosphate, 0.23 mM Nα-benzoyl-L-arginine ethyl ester (BAEE), 0.002 mM hydrochloric acid, 0.005mg trypsin, and 0.003 - 0.001 mg trypsin inhibitor.
Reagents Needed
S0751 - Sodium Phosphate monobasic
B4500 - Nα-Benzoyl-L-arginine ethyl ester hydrochloride (BAEE)
258148 - Hydrochloric acid ACS reagent
T8003 - Trypsin from bovine pancreas
Reagents
- 67 mM Sodium Phosphate Buffer, pH 7.6 at 25 °C
(Prepare 100 mL in deionized water using Sodium Phosphate, Monobasic, Anhydrous, Product No. S0751. Adjust to pH 7.6 at 25 °C with 1 M NaOH.) - 0.25 mM Nα-Benzoyl-L-Arginine Ethyl Ester Solution (BAEE)
(Prepare 50 mL in Reagent a using Nα-Benzoyl-L-Arginine Ethyl Ester, Hydrochloride, Product No. B4500.) - 1 mM Hydrochloric Acid Solution (HCl)
(Prepare 50 mL in deionized water using concentrated Hydrochloric Acid, Product No. 258148.) - Trypsin Enzyme Solution (Trypsin)
(Immediately before use, prepare a solution containing 1 mg protein/mL of Trypsin, Product No. T8003, in cold Reagent C.) - Trypsin Inhibitor Solution (Inhib.)
(Immediately before use, prepare a solution containing 1.0 mg/mL of Trypsin Inhibitor in cold Reagent A.)
Procedure
Pipette (in milliliters) the following reagents into suitable quartz cuvettes
Part A:
Uninh | Test 1 | Test 2 | Test 3 | Test 4 | Test 5 | |
---|---|---|---|---|---|---|
Reagent e (Inhib.) | — | 0.10 | 0.15 | 0.20 | 0.25 | 0.30 |
Reagent d (Trypsin) | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 |
Reagent c (HCl) | 9.50 | 9.40 | 9.35 | 9.30 | 9.25 | 9.20 |
Allow to stand at 25 °C for a minimum of five minutes and no longer than six minutes.
Mix by inversion and pipette (in milliliters) the following reagents into suitable cuvettes:
Part B:
Uninh | Test 1 | Test 2 | Test 3 | Test 4 | Test 5 | Blank | |
---|---|---|---|---|---|---|---|
Reagent b (BAEE) | 3.00 | 3.00 | 3.00 | 3.00 | 3.00 | 3.00 | 3.00 |
Reagent c (HCl) | 0.10 | 0.10 | 0.10 | 0.10 | 0.10 | 0.10 | 0.20 |
Mix by inversion and equilibrate to 25 °C. Monitor the A253nm until constant, using a suitably thermostatted spectrophotometer. Then add:
Uninh (Part A) | 0.10 | — | — | — | — | — | — |
Test 1 (Part A) | — | 0.10 | — | — | — | — | — |
Test 2 (Part A) | — | — | 0.10 | — | — | — | — |
Test 3 (Part A) | — | — | — | 0.10 | — | — | — |
Test 4 (Part A) | — | — | — | — | 0.10 | — | — |
Test 5 (Part A) | — | — | — | — | — | 0.10 | — |
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 Tests, Blank, and Uninhibited Solution.
Calculation
Trypsin Activity in BAEE units/mL enzyme = | (ΔA253nm/min Test – ΔA253nm/min Blank)(df)(10.0) (0.001)(0.10)(0.5) |
df = Dilution factor
0.001 = The change in A253nm/minute per unit of Trypsin at pH 7.6 at 25 °C in a 3.2 ml reaction mix
0.10 = Volume (in milliliters) enzyme used (Part B)
10.0 = Total volume in milliliters of assay (Part A)
0.5 = Volume (in milliliters) of enzyme used (Part A)
Units/mg solid = |
mg solid/mL enzyme |
Plot the Trypsin activity (BAEE units/mg protein) vs mL of Trypsin Inhibitor/RM
Mg Trypsin Inhibitor = (mL of Trypsin Inhibitor)(Conc. of Trypsin Inhibitor, mg/mL)
Mg Trypsin Inhibited by 1 mg Trypsin Inhibitor = |
mg Trypsin/RM (normalizing factor) mg Trypsin Inhibitor (from plot) |
Normalizing Factor = (BAEE Units of Uninhibited Trypsin per mg solid/10,000 BAEE units of Trypsin per specification.)
Notes:
- This enzyme assay is used to assay product numbers: T9003, T9008, T9128, T9253, T2011, T4385, T9378, and T0256.
- When assaying Trypsin Inhibitor, Type II-S, product number T9128, prepare a solution containing 0.60 mg/ml of Trypsin Inhibitor in cold Reagent a.
- The uninhibited Trypsin activity should be within 85% of the release value for activity.
- With 11,700 to 13,005 Trypsin units/mg solid per label, the acceptable range for activity of the uninhibited Trypsin reaction should be 10,000 to 15,300 Trypsin units/mg solid. This range should also correspond to a corrected ΔAbs253nm/minute of 0.0545 to 0.0835. With this rate and an inhibition of 20% to 80% the ΔAbs253nm/minute should be above the spectrophotometer rate detection limit of 0.0020.
Trypsin Unit Conversions
1 BAEE µM Unit = 200 BAEE Units
1 TAME µM Unit = 0.27 BAEE µM Units
1 BAEE µM Unit = 3.64 TAME Units
1 TAME µM Unit = 55 BAEE A253 Units
1 BAEE A253 Unit = 0.018 TAME µM Unit
1 TAME µM Unit = 180 TAME A247 Units
1 TAME A247 Unit = 0.33 BAEE Units
1 USP Unit = 3.0 BAEE Units
1 NF Unit = 1.1 USP Units
References
Natural trypsin Inhibitors also known as serine protease inhibitors (serpins) are the largest and most diverse family of protease inhibitors.1 Serpins control the activation and catabolism of proteins by the inhibition of serine proteases in vivo.2
There are four natural sources of trypsin inhibitors: bovine pancreas, ovomucoid, soybean, and lima bean. Each inhibitor acts as a competitive substrate analog and binds with its serine protease to form an inactive complex, therefore rendering the protease inactive.3
This process allows the serpin (trypsin inhibitor) to stop the proteolytic activity of the serine protease when its function is no longer necessary.
Trypsin inhibitors provide unique processes depending on their source. For example, inhibitors in the seeds of legumes (soybean and lima bean) act as a feeding deterrent for insects by disrupting midgut proteases. This natural function is being expanded upon in the development of insect resistant transgenic plants. Soybean inhibitors have also been found to contribute to pancreatic hypertrophy in rats, again providing a feeding deterrent. The Bowman-Birk soybean inhibitor is being studied as a cancer preventive agent.4
Products
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