ASSAY METHOD OF

NEUTRAL PROTEASE ACTIVITY

Date of Revision: 14/12/2017 In compliance with National Standard GB 1886.174

1. Definition of Enzyme Activity Unit

One Unit of enzyme activity of protease, expressed in U/g (or U/mL), is defined as one μg tyrosine
released from casein per minute during proteolysis by one gram protease powder (or one milliliter
protease liquid) under specific temperature and pH conditions.

2. Principle of Folin’s method

Protease hydrolyses casein substrate and releases amino acid that contains phenolic groups (such
as tyrosine and tryptophan) under specific temperature and pH conditions, which react with Folin’s
reagent under alkaline conditions to produce molybdenum blue and tungsten blue. Use
spectrophotometer to measure the absorbance of the solution at wavelength 680nm. Enzyme
activity is proportional to the absorbance, thus can be calculated.

3. Instrument and Equipment

3.1 Analysis balance: Accuracy 0.0001 gram
3.2 UV - visible spectrophotometer
3.3 Constant temperature water bath: Accuracy ±0.2°C
3.4 pH meter: Accuracy 0.01

4. Reagents and solutions

Unless otherwise indicated, the reagents used in this test is analytical grade, the water used in
this test is distilled-water or DI water.

4.1 Preparation of Folin’s reagents

Add 100.0g sodium tungstate (Na2WO4·2H2O), 25g sodium molybdate (Na2MoO4·2H2O),
700mL water, 50mL 85% phosphoric acid and 100mL concentrated hydrochloric acid into a
2000ml flask of mouth grinding backflow device. Boil on a small fire for 10h. Remove the back
cooler from the said device and put the same flask in a fume hood. Add 50g sulfuric acid lithium
(Li2SO4), 50mL water and several drops of concentrated bromine (99%) into the flask; Heat to
slightly boiling for 15min so as to remove the excessive bromine. (If the solution is still greenish
when it is cooled down, add another several drops of concentrated bromine and heat the solution
again to slightly boiling). Cool it down, add water into the flask to make the volume to 1000mL.
Mix evenly, filter. The prepared reagent should be golden brown and stored in a brown bottle.

4.2 Folin’s solution

Mix Folin reagent and water at a ratio of 1:2, shake even.

4.3 Sodium carbonate solution(42.4g/L)

Weigh 42.4g anhydrous sodium carbonate (Na2CO3), dissolve it with water and volume to 1000mL.

4.4 Trichloroacetic acid (65.4g / L)

Weigh 65.4g trichloroacetic acid(CCl3COOH), dissolve it with water and volume to 1000mL.

4.5 Sodium hydroxide solution (20g/L)

Weigh 20.0g sodium hydroxide (NaOH), add 900mL water and stir it until dissolved completely.
When the solution is cooled down to room temperature, make it to 1000mL with water and stir evenly.

4.6 Hydrochloric acid solution, c (HCl) = 1mol / L

4.7 Hydrochloric acid solution, c (HCl) = 0.1mol / L

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4.8 Buffer solution
Phosphate buffer (pH = 7.5)
Weigh 6.02g disodium hydrogen phosphate (Na2HPO4·12H2O) and 0.5g sodium
dihydrogen phosphate (NaH2PO4·2H2O) respectively, dissolve with water and volume to
1000mL
Calibrate with pH meter.

4.9 Casein solution (10.0 g /L )

Weigh NICPBP (National Institute for the Control of Pharmaceutical and Biological Products)
standard casein 1.000g, accurate to 0.001g; moisture it with small amount of sodium hydroxide ,
add 80mL of the readily prepared phosphate buffer solution , heat in a boiling water bath for 30
minutes and stir it during the heating process until the casein is completely dissolved. When the
casein solution is cooled down to room temperature, move it into a 100mL flask, dilute it to the
scale with buffer solution. This solution should be stored in the refrigerator with a validity of three
days. Before use, re-check the pH to specified value.
Note: Casein from different sources or batches has an effect on the test results. If different casein is
used as the substrate for this test, the results should be compared against the above-mentioned
standard casein before use.

4.10 L-tyrosine stand-by solution (100μg/mL )

Weigh 0.1000g ±0.0002g of L-tyrosine which is readily prepared at 105°C to constant weight,
dissolve it with 60mL 1mol / L hydrochloric acid, volume to 100mL, and gets the standard
1mg/mL tyrosine solution.
Pipet 10.00mL of the 1mg/mL tyrosine solution, volume to 100mL with 0.1mol /L hydrochloric
acid, and gets the 100μg/mL L-tyrosine stand-by solution.

5. Assay Procedure

5.1 Standard Curve

a) L-tyrosine standard solution: to be prepared according to Table 1.

Table 1 Preparations of L-tyrosine Standard Solution
Tube Concentration of
No. tyrosine standard solution
(µg/mL)
0 0
1 10
2 20
3 30
4 40
5 50
Volume of Volume of water
tyrosine stand-by solution to be added
(mL) (mL)
0 10
1 9
2 8
3 7
4 6
5 5

b) Pipet 1.00mL of the above mentioned solution separately (parallel test should be done), add
5.00mL sodium carbonate solution, 1.00mL Folin’s reagent solution, shake and mix evenly,
place it to 30 ±0.2°C water bath for 20 minutes, take out from the bath, measure absorbance
with the spectrophotometer using 10mm cuvette under wavelength 680nm, tube No. 0 ( does not
contain tyrosine) is measured as blank tube. Use absorbances (A) as the vertical axis, the
concentration of tyrosine (C) as abscissa, draw the standard curve.

c) Calculate the amount of tyrosine (μg) when the absorbance is 1 according to the regression
equation, which is the absorption constant K value. The K value should be with the range from 95
to 100.
Note: The L-tyrosine diluted solution must be used for assay immediately after dilution.

5.2 Preparation of enzyme solution

Weigh 1∼2 g enzyme powder (or pipette suck 1.00mL enzyme liquid) with an accuracy of
0.0002g, dissolve it with buffer solution and dilute it to appropriate concentration. Recommended
concentration is 10 U/mL∼15 U/mL enzymatic activity.

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5.3 Determination

First, put casein solution into 30 ± 0.2°C constant temperature water bath, warm-up for 5 minutes;
Second, conduct the following procedure:

Tube A (blank) Tube B

(enzyme samples, need to make three parallel
samples)
↓ ↓
1.00mL Enzyme solution 1.00mL enzyme solution
↓ 30 ±0.2°C, 2min ↓ 30 ±0.2°C, 2min

Add 2.00mL trichloroacetic acid (shake evenly) Add 1.00mL casein solution (shake evenly)
↓ 30 ±0.2°C, 10min ↓ 30 ±0.2°C, 10min
Add 1.00mL casein (shake even) Add 2.00mL trichloroacetic acid (shake even)
↓ ↓
Take out of the bath and standstill for 10min, Take out of the bath and standstill for 10min,
filter with slow speed qualitative filter paper filter with slow speed qualitative filter paper
↓ ↓
Pipette 1.00mL filtrate Pipette 1.00mL filtrate
↓ ↓
Add 5.0mL sodium carbonate solution Add 5.0mL sodium carbonate solution
↓ ↓
Add 1.00mL Fulin reagent solution Add 1.00mL Fulin reagent solution
↓ 30 ±0.2 °C, chromogenic for 20min ↓ 30 ± 0.2°C, chromogenic for 20min
Measure absorbance with 10mm cuvette Measure absorbance with 10mm cuvette
under 680nm wavelength. under 680nm wavelength.

5.4 Calculation

X = A × K × 4 / 10 × n = 2 / 5 × A × K × n

In this formula:
X------Enzyme activity of the sample, u / g (u / mL)
A------Average absorbance of the parallel tests
K------Absorption constant
4------Total volume of reaction reagents, mL
10-----Reaction time 10 minutes, calculated by 1 minute
n------Dilution times

The calculation results should be expressed as integer.

5.5 Tolerance of the testing results

Relative error of the parallel tests should not exceed 3%

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