Protease activity assay by modified Anson method

I. Introduction
Widely used in food and pharmaceutical industries, proteases represent the largest segment
of the industrial enzyme market. Theses enzymes catalyze the hydrolysis of the peptide
bond and therefore break proteins into small fragments. In this procedure, protease activity
is determined by the modified Anson method using casein as a substrate. Under the action
of the protease, the substrate casein is hydrolyzed to release tyrosine and tryptophan along
with other amino acids and peptide fragments. After the removal of undigested protein by
trichloroacetic acid (TCA), Folin’s reagent is added to the hydrolysate to react with
tyrosine and tryptophan to produce a blue colored chromophore, which is quantifiable and
measured as an absorbance value on the spectrophotometer. From the standard curve
depicting the relationship between tyrosine concentration and OD750nm after reacting with
Folin’s reagent, the activity of protease samples can be determined in terms of Units, which
is the amount in micromoles of tyrosine equivalents released from casein per minute.

II. Materials
- Substrate solution: 1% casein in phosphate buffer, pH 7.0
- Diluted enzyme solution in phosphate buffer pH 7.0
- 0.3 M Trichloroacetic acid (TCA) solution
- 0.5 M Na2CO3 solution
- Water bath
- Filter paper, micropipettes, glass tubes

III. Procedure
- Pre-equilibrate the substrate solution and the enzyme solution at 30°C for at
least 5 min.
 - Pipette the following reagent into glass tubes
Test sample Blank sample
- 2 mL of substrate solution. - 2 mL of casein solution.
Step 1.
- 2 ml of enzyme solution. - Incubate for exactly 10 minutes at
Enzyme
- Mix well. 30°C.
reaction
- Incubate for exactly 10 minutes at 30°C
Step 2.
- Add 4 mL of 0.3 M TCA.
Enzyme - Mix well.
- Add 4 mL of 0.3 M TCA.
inactivation - Add 2 mL of enzyme solution.
- Mix well immediately.
and - Mix well immediately.
- Incubate for 10 minutes at 30°C.
substrate - Incubate for 10 minutes at 30°C.
- Filter, collect the filtrate.
removal by - Filter, collect the filtrate.
TCA
- Add 0.3 mL of the filtrate to a new - Add 0.3 mL of the filtrate to a new
tube. tube.
Step 3.
- Add 1.5 mL of 0.5 M Na2CO3. - Add 1.5 mL of 0.5 M Na2CO3.
Reaction - Mix well, incubate for 10 minutes.
- Mix well, incubate for 10 minutes.
with Folin’s - Add 0.3 mL of Folin’s reagent.
- Add 0.3 mL of Folin’s reagent.
reagent - Mix well.
- Mix well.
- Incubate for 30 minutes. - Incubate for 30 minutes.
Step 4.
- Measure the absorbance at 750 nm (use the blank sample to set the
Absorbance
spectrophotometer to zero).
measurement

IV. Calculation

Calculate the protease activity (U/mL) using the standard curve provided by the laboratory.

Note: One unit of protease is the amount of enzyme that liberates TCA-soluble product
equivalent to 1 µmol tyrosine within one minute at 30°C using casein as a substrate.