Assay Procedure for Protease

Principle

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

Unit definition

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

Method

Reagents

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

Procedure

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

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

2. Add 0.5 mL of the enzyme solution* and mix.
   
3. After exactly 10 minutes at 30 ℃, add 3.2 mL 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 275 nm (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 10 mM 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 (6.7 mL)
Vs	：Sample volume (0.5 mL)
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.