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