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Trypsin Inhibitors - Assay
CAS:
9035-81-1
Trypsin Inhibitors Assay
Method
The ability of the various trypsin inhibitors to prevent trypsin hydrolysis of benzoyl-L-arginine ethyl ester is measured spectrophotometrically. No unit of activity is currently assigned. The activity of the inhibitors is expressed as the amount of twice crystallized trypsin (TRL) inhibited by one milligram of inhibitor.
Reagents
- Trypsin reagent: 0.25 mM benzoyl-L-arginine ethyl ester in 0.067 M phosphate buffer, pH 7.0
- 0.001 N Hydrochloric acid
- 0.5 M Sodium potassium phosphate buffer, pH 6.5
- Trypsin solution: Prepare trypsin (Worthington Code: TRL) at 0.5 mg/ml in 0.001 N HCl.
- Inhibitors: Dissolve various inhibitors as follows:
Lima bean inhibitor: 1 mg/ml in 1% NaCl Ovomucoid: 1 mg/ml in 0.001 N HCl Pancreatic inhibitor: 1 mg/ml in 0.001 N HCl Soybean inhibitor: 1 mg/ml in 0.01 M phosphate
buffer, pH 6.5 with 0.15M NaCl
Procedure
Pipette the following into a series of test tubes:
Trypsin solution 1.0 ml 0.5 M Phosphate buffer 2.0 ml Inhibitor * Reagent grade water q.s. to 10 ml
Include 2 test tubes with no inhibitor as controls.
* The following amounts are recommended:
Lima bean inhibitor: 50 - 300 micrograms Ovomucoid and purified soybean inhibitor: 100 - 500 micrograms Pancreatic inhibitor: 20 - 500 micrograms Crude soybean inhibitor : 200 - 1000 micrograms
Incubate tubes at room temperature for 30 minutes. Dilute an aliquot with an equal portion of water and assay for residual trypsin activity. Set spectrophotometer at 25°C and 253 nm. Pipette 2.9 ml of trypsin reagent into cuvette and incubate in spectrophotometer for 3-4 minutes to achieve temperature equilibration and establish blank rate, if any. Add 0.1 ml diluted incubation mixture and record increase in A253 for 3-4 minutes. Determine ΔA253/minute from initial linear portion of curve.
Calculation
Trypsin Inhibitors Products
Description
Activity
Code
Cat. #
Size
Price
Description
Trypsin Inhibitor, Lima Bean, AF
Source:
Lima Bean
Animal Free/AF. Fraction III of the preparation described by Fraenkel-Conrat, et al., Arch. Biochem. Biophys., 37, 393 (1952). Supplied as a dialyzed, lyophilized powder.
Store at 2-8°C.
Code
LBI
Product details
LS002829
100 mg
$160.00
LS002830
1 gm
$1,150.00
LS002831
Bulk
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Description
Trypsin Inhbitor, Ovomucoid
Source:
Egg White
Mucoprotein and antitryptic factor of egg white described by Lineweaver and Murray, J. Biol. Chem., 171, 565 (1947). A dialyzed, dried powder.
Store at 2-8°C.
Code
OI
Product details
LS003085
500 mg
$94.00
LS003087
1 gm
$160.00
LS003086
2 gm
$310.00
LS003089
Bulk
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Description
Trypsin Inhibitor, Soybean, Purified, AF
Source:
Soybean
Chromatographically purified as described by Frattali, V., and Steiner, R.: Biochem., 7, 521 (1968), modified by Worthington. A dialyzed, lyophilized powder. Purity >90% checked using SDS PAGE.
Store at 2-8°C.
Code
SI
Product details
LS003570
100 mg
$80.00
LS003571
1 gm
$500.00
LS003573
Bulk
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Description
Trypsin Inhibitor, Soybean, AF
Source:
Soybean
A partially purified lyophilized powder.
Store at 2-8°C.
Code
SIC
Product details
LS003589
10 gm
$700.00
LS003587
1 gm
$92.00
LS003590
Bulk
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