METHOD 5:2

Printed with effect from 1st January 2003

DIGESTIBLE CRUDE PROTEIN PEPSIN ACTIVITY

1: Scope and Field of Application

This method is for the determination of the activity of pepsin used in the determination of
crude proteins soluble in pepsin and hydrochloric acid in feeding stuffs (GAFTA
Method 5:1).

2: Principle
Haemoglobin is treated with pepsin in a hydrochloric acid medium under controlled
conditions. The non-hydrolysed protein is precipitated by trichloroacetic acid solution and
filtered. Sodium hydroxide solution and Folin-Ciocalteu's reagent are added to the filtrate
and the absorbance of the solution measured at 750nm. The corresponding quantity of
tyrosine is then read from a calibration curve.

NOTE: the unit of pepsin activity is defined as being that quantity of the enzyme which
liberates per minute, under the prescribed conditions, a quantity of hydroxyaryl groups
whose coloration by Folin-Ciocalteu's reagent has an absorbance corresponding to that
given by 1µ mole of tyrosine under the same conditions.

3: Reagents
3:1 Trichloroacetic acid solution: 5g trichloroacetic acid per 100ml.
3:2 Sodium hydroxide solution, 0.5N.
3:3 Hydrochloric acid solution, 0.025N.
3:4 Hydrochloric acid solution, 0.06N.
3:5 Hydrochloric acid solution, 0.2N.
3:6 Folin-Ciocalteu's reagent: dissolve 100g sodium tungstate dihydrate and 25g sodium
molybdate dihydrate in 700ml water contained in a 2,000ml round-bottomed flask fitted
with a reflux condenser. Add 50ml phosphoric acid (d=1.75g/ml) and 100ml hydrochloric
acid (d=1.18g/ml) and boil gently under reflux for 10 hours. Cool, remove the reflux
condenser and add 175g lithium sulphate dihydrate, 50ml water and 1ml bromine. Boil
for 15 minutes to eliminate the excess bromine. Cool and transfer the solution to a 1 litre
graduated flask, dilute to volume with water, mix and filter. The resulting solution should
be free of any green colour. Dilute one volume of this reagent with two volumes of water
before use.
3:7 Haemoglobin solution: determine the nitrogen content of the haemoglobin by Kjeldahl's
method (GAFTA Method 4) and weigh to the nearest 0.001g a quantity corresponding to
354mg nitrogen. Transfer to a 200ml graduated flask provided with a ground-glass joint,
add a few ml 0.06N hydrochloric acid (3.4) and connect to a vacuum pump. Apply the
vacuum and shake the flask until the haemoglobin has completely dissolved. Release the
vacuum and, whilst shaking the flask continuously, dilute to volume with 0.06N
hydrochloric acid solution (3.4). Prepare the solution immediately before use.

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3:8 Standard solution of tyrosine: dissolve 181.2mg of tyrosine in 0.2N hydrochloric acid
solution (3.5) and dilute to 1,000ml with the same acid. Transfer by pipette 20ml of this
solution to a 100ml graduated flask and make up to the mark with 0.2N hydrochloric acid
(3.5). 1ml of this solution contains 0.2µmole tyrosine.

4: Apparatus
4:1 Water bath capable of being maintained at 25+1oC.
4:2 Spectrophotometer with 10mm cells.
4:3 Precision chronometer, accurate to 1 second.
4:4 pH meter.

5: Procedure
5:1 Preparation of pepsin solution
Dissolve 150mg pepsin in 100ml 0.06N hydrochloric acid (3.4) and transfer by pipette
2ml of this solution into a 50ml graduated flask. Dilute to volume with 0.025N
hydrochloric acid (3.3). Using the pH meter (4.4), check that the pH is 1.6+0.1, then
immerse the flask in the water bath maintained at 25oC (4.1).

5:2 Hydrolysis
Transfer by pipette 5ml haemoglobin solution (3.7) to a test tube and heat to a temperature
of 25oC in the water bath (4.1). Add 1.0ml pepsin solution (5.1) and mix thoroughly.
Keep the test tube in the water bath for exactly 10 minutes, timed from the addition of the
pepsin solution. (Both the timing and the water bath temperature are critical and must be
carefully observed). Add 10.0ml trichloroacetic acid solution (3.1) previously heated to
25oC, and mix and filter through a dry filter.

5:3 Colour development and measurement of absorbance

Transfer by pipette 5ml of the filtrate (5.2) to a 50ml Erlenmeyer flask, add 10ml 0.5N
sodium hydroxide solution (3.2) and, shaking the flask continuously, 3.0ml of diluted
Folin-Ciocalteu's reagent (3.6). Measure the absorbance of the solution at 750nm in the
spectrophotometer (4.2) after 5 to 10 minutes, using water as a reference.

5:4 Blank test

For each determination prepare a blank as follows:
Transfer by pipette 5ml of the solution, obtained in 3.7 into a test tube, heat to 25oC in a
water bath (4.1) and add 10.0ml trichloroacetic acid solution (3.1) previously heated to
25oC, mix and then add 1.0ml of pepsin solution (5.1). Mix thoroughly and place the flask
in a water bath maintained at 25oC for exactly 10 minutes. Mix again and filter through a
dry filter. Continue as in 5.3 from.... Transfer by pipette 5ml of the filtrate....

5:5 Calibration curve

Transfer into a series of 50ml Erlenmeyer flasks 1.0, 2.0, 3.0, 4.0, and 5.0ml aliquot parts
of standard tyrosine solution (3.8). The flasks contain 0.2, 0.4, 0.6, 0.8 and 1.0µ moles
tyrosine respectively. Complete the series with a flask containing no tyrosine as blank.
Make up the volume in each flask to 5.0ml with 0.2N hydrochloric acid (3.5). Add 10.0ml
0.5N sodium hydroxide solution (3.2), and shaking continuously, add 3.0ml of diluted
Folin-Ciocalteu's reagent (3.6). Measure the absorbances of the solutions as described in
the last sentence of 5.3. Construct a graph relating absorbances to the amounts of tyrosine

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 present.

6: Expression of the Results

By reference to the calibration curve obtained in 5.5, determine the quantity of tyrosine, in
µ moles, corresponding to the absorbance of the sample solution and corrected for the
blank test.
The pepsin activity, in µ moles of tyrosine per mg per minute at 25oC is given by the
formula:
Units per mg (U/mg)=0.32a
p
where:
a= quantity of tyrosine in µ moles, determined in the sample solution; and
p= weight of pepsin (in milligrams) added in 5.2.
NOTE 1: The quantity of pepsin taken for the preparation of the pepsin solution in 5.1 must
be adjusted in order to obtain an absorbance of 0.35+0.035 after 5 to 10 minutes.

2 units per mg obtained by this method correspond to:

3.64 milliunits Anson/mg (µ mole of tyrosine/mg/min at 35.5oC) or
36,400 commercial units/g (µ moles of tyrosine/g in 10 minutes at 35.5oC).
NOTE 2: For further information on the preparation of haemaglobin see Anson M.L., J.
Gen. Physiol., 1938, 22, 79.

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