Data Sheet - Trypsin

Bovine or porcine pancreas
Systematic name:
Peptidyl peptide hydrolase;


Trypsin is a proteolytic enzyme found in many animals and even bacteria. In vertebrates an inactive precursor, the Trypsinogen, is formed in the pancreas. By the action of Enterokinase or of Trypsin itself, Trypsinogen is transformed into active Trypsin.


Specificity: Trypsin hydrolyzes peptides, amides and esters at bonds involving the carboxyl group of L-arginine and L-lysine. Trypsin acts on numerous synthetic substrates, the best being esters of L-arginine, e.g. N-benzoyl-L-arginine ethyl ester. Proteins are only slowly degraded unless denatured (1).

Effectors: In the process of Trypsinogen activation Ca2+ ions have an accelerating effect and they are also essential for complete activation (1,2,3).

Trypsin is inhibited by a wide variety of substances. Trypsin is a serine proteinase (EC 3.4.21) and is therefore inhibited by compounds such as phenylmethyl-sulfonylfluoride (PMSF) and diisopropylphosphofluoridate (DFP). Aromatic and aliphatic amidines and amines are also inhibitory, the strongest low molecular weight competitive inhibitor of Trypsin being p-aminobenzamidine (1,4). Tosyl-L-lysine chloromethyl ketone (TLCK) irreversibly inhibits Trypsin (but not Chymotrypsin) (1,2,5).

High molecular weight inhibitors of Trypsin comprise polypeptides and proteins from animal origin (aprotinin, a2-macroglobulin, a1-antitrypsin, bovine pancreatic Trypsin inhibitor etc., as well as those isolated from plants e.g. from soybean, lima bean, and barley (see also: Methods in Enzymology, Vol. XIX, pp. 840-915).

Catalytic optima: Trypsin acts optimally at pH values between 7 and 9 (1,6).

Stability: Trypsin is most stable at pH 3. At this pH at low temperature it retains its activity for weeks (1,2). Autolysis of Trypsin at pH values well above 3 can be retarded by the addition of calcium ions. This protective effect is much more pronounced with bovine Trypsin than with porcine Trypsin (2). Heat denaturation is dependent on the pH: below pH 8 an increased temperature leads to reversible denaturation, but above pH 8 a temperature rise induces irreversible denaturation (2,3). Precipitation with trichloroacetic acid or the presence of high concentrations of urea (8 M) leads to a reversible loss of Trypsin activity (1). Lyophilized Trypsin, when stored cool and dry in the dark, is stable almost indefinitely (7).

Solubility: Trypsin is soluble in water and in isotonic saline solution.

Molecular weight: approx. 23,500 (4).

Composition: Bovine Trypsinogen consists of a single polypeptide chain with 229 amino acid residues stabilized by 6 disulfide bridges. Upon activation, a hexapeptide (Val-(Asp)4-Lys-) is split off from the amino terminus leading to the formation of b-Trypsin, the first active enzyme form. Sequential cleavage at two other sites leads to a-Trypsin, and Pseudotrypsin, respectively, both of which are also active forms of Trypsin (1,2,3).

Isoelectric point: 10.8 (bovine and porcine Trypsin) (1,3).

Spectral data: E280 (1%, 1cm) = 15.4 (1)

Assay (according to current FIP-Method)

The method described here is the one given in the monography on Trypsin in the Ph.Eur. is identical to the method given by FIP (Fédération International Pharmaceutique; see 3) which originally developed this assay method.


Trypsin hydrolyzes the synthetic substrate N-benzoyl-L-arginine ethyl ester (BAEE). The amount of acid liberated (at pH 8.0 and 25 °C) is measured by titration with sodium hydroxide, recorded as a function of time.

The activity of Trypsin is determined by comparing the rate at which it hydrolyzes benzoylarginine ethyl ester with the rate at which Trypsin BRP hydrolyzes the same substrate under the same conditions.


Use a reaction vessel of about 30 ml capacity provided with:

An automatic or manual titration apparatus may be used. For the latter, the burette is graduated in 0.005 ml and the pH meter is provided with a wide scale and glass-calomel electrodes.


  1. BAEE solution (0.02 M): 0.686 % (m/V). Dissolve 0.1714 g of N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE x HCl) in distilled water and dilute to 25.0 ml with the same solvent.
  2. Borate buffer solution pH 8.0 (0.0015 M): Dissolve 0.572 g of disodium tetraborate (Na2B4O7 x 10 H2O) and 2.94 g of calcium chloride (CaCl2 x 2 H2O) in 800 ml of water. Adjust with 1 N hydrochloric acid to pH 8.0 and dilute to 1000.0 ml with water.
  3. 0.1 N Sodium hydroxide.
  4. 0.001 N Hydrochloric acid.
  5. Test solution: Dissolve sufficient of the substance to be examined in 0.001 N hydrochloric acid (D) and dilute to 25.0 ml with the same acid in order to obtain a solution containing approximately 700 nanokatals per ml.
  6. Reference solution: Dissolve 25.0 mg of Trypsin BRP in 0.001 N hydrochloric acid (D) and dilute to 25.0 ml with the same acid.

Trypsin BRP is issued by: Technical Secretariat, European Pharmacopoeia Commission, Council of Europe, P.O. Box 907 R6, 67029 Strasbourg CEDEX 1, France.


Store the test solution (E) and the reference solution (F) at 0 °C to 5 °C. Warm 1 ml of each solution to about 25 °C over 15 min and use 50 ml of each solution for each titration. Carry out the titration in an atmosphere of nitrogen.

Transfer into the reaction vessel 10.0 ml of borate buffer solution pH 8.0 (B) and add, while stirring 1.0 ml of BAEE solution (0.02 M) (A).
When the temperature is steady at 25.0 ± 0.1 °C, adjust the pH to exactly 8.0 with 0.1 N sodium hydroxide (C), then add 50 ml of the test solution (E), start stop-watch and maintain the pH at 8.0 by the addition of 0.1 N sodium hydroxide (C), the tip of the microburette being immersed in the solution. Note the volume added every 30 s.

Follow the reaction for 8 min. Calculate the volume of 0.1 N sodium hydroxide used per second. Carry out a titration in the same manner using the reference solution and calculate the volume of 0.1 N sodium hydroxide used per second.

Calculate the activity in microkatals per milligram using the expression:

m' x  V
x A
m  x  V'

m    =   mass in milligrams of the substance to be examined,
m'    =   mass in milligrams of Trypsin BRP,
V    =   volume of 0.1 N sodium hydroxide used per second by the test solution,
V'    =   volume of 0.1 N sodium hydroxide used per second by the reference solution,
A    =   activity of Trypsin BRP in microkatals per milligram.

Other assays

In the FIP assay for Trypsin (see above) the specific activity is given in FIP units/mg. 0.5 µkatal/mg = 30 FIP units/mg.
In the assay for Trypsin activity according to USP 29 the rate of hydrolysis of BAEE at pH 7.6 is measured spectrophotometrically at 253 nm.

Assay (according to USP-Monograph)

One USP Trypsin Unit is the activity which hydrolses 1 µmol Na- Benzoyl-L-argininethylesterhydrochlorid (= BAEE*HCl) such that causing a change in absorbance of 0.003 per minute under the conditions specified in this assay at 253 nm.


  1. Temperature equalisation: Trypsin Reference Standard USP, the Samples and BAEE*HCl (= Na- Benzoyl-L-argininethylesterhydrochlorid) from 4 °C to Room temperature.
  2. 1 mM HCl-Solution: 1.0 ml 1 N HCl-Solution + pure H2O add. to 1000 ml.
  3. BAEE-Solution: 85.7 mg BAEE*HCl (M = 342.83 g/mol, Calbiochem 2645-08-1) + pure H2O add to 100.0 ml.
  4. 0.067 M KH2PO4-Solution: 4.56 g KH2PO4 (M=136.09 g/mol, MERCK 4873) + pure H2O add to 500 ml.
  5. 0.067 M Na2HPO4-Solution: 4,73 g Na2HPO4 (M = 141,96 g/mol, MERCK 6586) + pure H2O add to 500 ml.
  6. 0.067 M Phosphat-Buffer, pH 7.60: Give to 200 ml Na2HPO4-Solution an amount of KH2PO4 -Solution until it adjust a pH of 7.60.
  7. Enzyme-Solution: Test the Activity of each Sample with two Initial weight. Weight an amount of Enzyme Sample or Standard that solved and diluted in 1 mM HCl-Solution give a change of absorbance (=DA/min) of maximal 0.02 in „ml in Test“ of 0.200.


Initial weight (mg) =
[(5.0 USP-U / “0.200“ ml in Test) * dilution factor * Enzyme solution]
Declared activity of enzyme

Because in the most of cases the product is a mixture of Trypsin and Chymotrypsin the initial weight should be chosen such that you can use the same Enzyme solution for determining the activity of Trypsin and Chymotrypsin with a „ml in Test“ of 0.080-0.200 ml.


For each 90 ml of Phosphat-Buffer use 10 ml BAEE-Solution (1:10). The Absorbance should be at 253 nm 0.575 - 0.585. If necessary add more Phosphat-Buffer or BAEE-Solution.
Put the amount Phosphate-Buffer-Solution in Bath thermostat to adjust the temperature to 25 °C. Shortly before test begin give the necessary amount of BAEE-Solution. The Solution at 25 °C is stable for 2 h. To reach a homogeny temperature, mix the solution before using each time.

  1. Adjust the UV-Spectral photometer such that the change of absorbance for each reaction in the cuvettes can be reading every 30 seconds for 5 minutes.
  2. Calibrate the UV-Spectral photometer at 253 nm using pure H2O.
  3. Blank: Give in a cuvette 200 µl of 1 mM HCl-Solution and 3 ml of Phosphate-Buffer-Solution and mix. Calibrate the UV-Spectral photometer with this solution.
  4. After calibration give in the cuvette e.g. 100 µl of Enzyme dilution, 100 µl of 1 mM HCl-Solution and 3 ml of Phosphate-Buffer-Solution. Mix immediately put in UV-Spectral photometer and start reading the absorbance.

    Is the Change of Absorbance (= Δ A/min) higher than 0.02 or lower than 0.009 then change for the next measurement the amount of “µl in Test” of 80-200 µl.

  5. Print the << Rates >> of absorbance and the tabulate of „Δ A/min“.
  6. Prepare the UV-Spectral photometer according the manufacturer instruction to starting a new measurement.
  7. Measure each Enzyme dilution 4 times.
  8. Clear the cuvette for each using with pure H2O.


Calculate the average of Δ A/min of each Enzyme-Solution.

Factor =
{[(Initial weight (mg)/Enzyme solution (ml))/(dilution factor)]*(ml in Test)]} * 0.003

USP-unit/mg (abs.) = [Δ A/min]* Factor
Standard: USP-unit/mg (rel.) = r = USP-unit/mg (abs.) / declared activity of reference Standard
Sample: USP-unit/mg (rel.) = USP-unit/mg (abs.) / r


Standard qualities
Trypsin 2500 USP units/mg
Trypsin 3200 USP units/mg
Trypsin 4500 USP units/mg
Trypsin Ph. Eur. (0.5 µkatal/mg)
Trypsin 30 FIP units/mg

Customized qualities are available upon request.


  1. Keil, B. in: The Enzymes (P.D. Boyer, ed.) 3rd ed., Vol. III, p. 250. Academic Press, New York, 1971.
  2. Walsh, K.A. in: Methods in Enzymology (G.E. Perlmann, L. Lorand, eds.) Vol. XIX, p. 41. Academic Press, Inc., Orlando, Florida, 1970.
  3. Lauwers, A.; Scharpé, S.: Pharmaceutical Enzymes, drugs and pharmaceutical sciences., Volume 84, Marcel Dekker, Inc., New York-Basel-Hong Kong, 1997.
  4. Barman, T.E.: Enzyme Handbook, Vol. II. Springer, New York-Heidelberg-Tokyo, 1985.
  5. International Union of Biochemistry. Nomenclature Committee: Enzyme Nomenclature. Academic Press, Inc., London, 1984.
  6. Merck Index, 11th ed., Merck & Co., Inc., Rahway, USA, 1989.
  7. Stellmach, B.: Bestimmungsmethoden Enzyme. Steinkopff Verlag Darmstadt 1988.


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