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

A. Casein solution	：0.6% ［Suspend 3.0g of Hammersten milk casein in 20mL to 0.1N NaOH solution and dissolve by warming in a hot water bath (60 ℃). Cool down to room temperature, add 50mL of 0.1M borax and 400mL of H2O and, after adjusting the pH to 11.0 with 1.0N NaOH solution, fill up to 500mL with H2O］ (Stable for one week if stored at 0－5 ℃)
B. TCA mixture	：0.11M Trichloroacetic acid (TCA) containing 0.22M sodium acetate and 0.33M acetic acid
C. Enzyme diluent	：2.0mM calcium acetate

Procedure

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

Concentration in assay mixture
Borax	8.6 mM
Casein	5.1mg/mL
Ca(CH3COO)2	0.29 mM

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

Vt	：Total volume (6.7mL)
Vs	：Sample volume (0.5mL)
1.34	：Millimolar extinction coefficient of tyrosine (F/micromole)
1.0	：Light path length (cm)
t	：Reaction time (10 minutes)
df	：Dilution factor
C	：Enzyme concentration in dissolution (c mg/mL)

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