Assay Procedure for Protease

Principle

The appearance of peptides is measured as tyrosine equivalent at 275nm by spectrophotomertry.

Unit definition

One unit causes the increase of optical density at 275nm corresponding to one micromoloe of tyrosine per minute under the conditions described below.

Method

Reagents

Procedure

1. Pipette 3.0mL of substrate solution (A) into a test tube and equilibrate at 30 ℃ for about 5minutes.

2. Add 0.5mL of the enzyme solution* and mix.
   
3. After exactly 10 minutes at 30 ℃, add 3.2mL of TCA mixture (B) to stop the reaction.
   
4. Incubate for further 20 minutes at 30 ℃.
   
5. Filter the mixture with a filter paper (Toyo Roshi No.131) and measure the optical density of the filtrate at 275nm (OD test).
   

At the same time, prepare the blank by first mixing the substrate solution with 3.2mL of TCA mixture (B) after 10 min-incubation at 30 ℃, followed by addition of the enzyme solution, and carry out the same procedure (procedure 4－5) at test (OD blank).

* Dissolve the enzyme preparation in ice-cold 10mM borax-NaOH buffer, pH 11.0 and dilute to 0.1－0.4U/mL with enzyme diluent (C), immediately before assay.

Calculation

Activity can be calculated by using the following formula：

Weight activity (U/mg)＝(U/mL)×1/C

This procedure is for informational purposes. For a current copy of our control procedure, please contact our Technical Service Department.