Specialists in the Isolation and Purification of Horseradish Peroxidase and a Select Range of Enzymes

Assay Procedure for Trypsin Inhibitor  (Ref. No. FGAP012)

Based on that of Laskowski which measures residual trypsin activity in a solution of known activity after addition of inhibitor.

Unit Definition

That amount of inhibitor causing the inhibition of one BAEE unit of trypsin. One BAEE trypsin unit is that amount of enzyme causing an absorbance increase at 253nm of 0.001 per minute at 25^°C.

Reagents

A	0.067M Potassium Phosphate Buffer pH7.6 Dissolve 0.59g KH2PO4 [MM 136.09] and 5.06g K2HPO4 [MM 174.18] in distilled H2O, check pH 7.6 and dilute to 500ml. Store on ice.
B	0.01M Borate Buffer pH9.0 Dissolve 0.309g H3BO3 [MM 61.83] in 400ml distilled H2O, adjust pH to 9.0 with 1M NaOH and dilute to 500 ml. Store on ice.
C	0.005M HCl Dilute 0.45ml concentrated HCl to 1000ml with distilled H2O. Store on ice.
D	Substrate [0.00025M N-alpha-Benzoyl-L-Arginine Ethyl Ester] Dissolve 8.6mg BAEE-HCl [MM342.83] / 100ml buffer [A] and adjust A253 to 0.575.
E	Inhibitor Dissolve 1mg inhibitor/ml ice-cold borate buffer [B]. Immediately before assay, dilute solution to yield 2000 BAEE units of inhibition/ml ice-cold buffer [B]. Dilution of inhibitor should inhibit 50% of trypsin.
F	Trypsin Dissolve 1mg international NF Standard or Internal House Standard / 1ml ice-cold HCl [C]. [1 NF unit = 3 BAEE units] BAEE units allocated to standard.           [A]

Procedure

a) Assay trypsin standard solution by pipetting 3.0ml substrate [D] and 0.2ml enzyme (trypsin) into a 10mm quartz cuvette. Record rate of increase of absorbance at 253nm for approximately three minutes (or until reaction runs to completion) and calculate activity (using linear portion of graph) as follows:

	DA253 / min x dilution	[B]
u/mg standard =
	0.001 x 0.2 x 1

b) Based on activity results of trypsin standard prepare a solution at 4000 BAEE units/ml ice-cold HCl [C].

c) In a test tube, mix 1ml ice-cold borate buffer [B] and 1ml trypsin [F] at 4000 BAEE units/ml. Allow to stand in ice for 15 minutes, dilute 1/16 to yield 120 BAEE units/ml HCl [C] and assay for trypsin as under (a).

	DA253 / min x 2 x 16	[C]
U/ml =
	0.001 x 0.2

d) In a test tube mix 1ml inhibitor [E] and 1ml trypsin [F] at 4000 BAEE u/ml, allow to stand in ice for 15 minutes, dilute solution 1/8 to yield 120 BAEE u/ml ice-cold HCl [C] and assay for trypsin as under (a).

	DA253 / min x 2 x 8	[D]
U/ml =
	0.001 x 0.2 x 1

Repeat with different dilution of inhibitor until about 50% inhibition is achieved.

DA₂₅₃ for (a), (c) and (d) should be approximately the same.

e) Correct values [C] and [D] by multiplying by [A] / [B] and calculate inhibitor activity as follows:

BAEE units of inhibition / mg inhibitor	[C] x [A]		- [D] x [A]		x inhibitor dilution
	[B]		[B]

Reference

Laskowski M.: (1955) Methods in Enzymology 2 37. Ed. By Colowick S.P. and Kaplan N.O. Academic Press, New York. Schwert C.W. and Takenaka Y.: (1955) Biochem. Biophys. Acta. 16 570.