Trypsin Inhibitors Assay

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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

\frac{\text{mg TRL inhibited}}{\text{mg inhibitor}}\text{ = }\frac{\underset{\text{Control}}{\text{(}\frac{\Delta \text{A}_{253}}{\text{min}}}\text{ - }\underset{\text{Sample}}{\frac{\Delta \text{A}_{253}}{\text{min}}\text{)}}\text{ x 5.0}}{\underset{\text{Control}}{\frac{\Delta \text{A}_{253}}{\text{min}}}\text{ x }\mu \text{g inhibitor in reaction mixture}}

Trypsin Inhibitors Products

Description

Trypsin Inhibitor, Lima Bean, Animal Free/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.

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Activity

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Certificate of Analysis

Code

LBI

Product details

LS002829

100 mg

$150.00

LS002830

1 gm

$1,100.00

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.

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Activity

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Certificate of Analysis

Code

Product details

LS003085

500 mg

$88.00

LS003087

1 gm

$150.00

LS003086

2 gm

$280.00

LS003089

Bulk

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Description

Trypsin Inhibitor, Soybean, Purified, Animal Free/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.

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Activity

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Certificate of Analysis

Code

Product details

LS003570

100 mg

$74.00

LS003571

1 gm

$458.00

LS003573

Bulk

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Description

Trypsin Inhibitor, Soybean, Animal Free/AF

Source:

Soybean

A partially purified lyophilized powder.

Store at 2-8°C.

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Activity

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Certificate of Analysis

Code

SIC

Product details

LS003589

10 gm

$660.00

LS003587

1 gm

$86.00

LS003590

Bulk

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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

Trypsin solution	1.0 ml
0.5 M Phosphate buffer	2.0 ml
Inhibitor	*
Reagent grade water	q.s. to 10 ml

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

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Trypsin Inhibitors - Assay

Trypsin Inhibitors Assay

Trypsin Inhibitors Products