Trypsin

Physical Properties and In Vivo Processing

Trypsin
Enzyme Commission (EC) Number: 3.4.21.4
Molecular Weight: 23.3 kDa^1,2 (bovine & porcine)
Extinction Coefficient: E^1% = 12.9 - 15.4 (280 nm)^3,4
pI: 10.1 - 10.5^2,5(bovine)

Trypsinogen
Molecular Weight: 24 kDa⁵ (bovine)
Extinction Coefficient: E^1% = 14.4 (280 nm)
pI: 9.3² (bovine)

Trypsinogen, the proenzyme (zymogen) form of trypsin, is produced in the acinar exocrine cells of the pancreas. Three isoforms are excreted from the human pancreas. The cationic and anionic forms are the predominant human isoforms. The inhibitor-resistant mesotrypsinogen is found only in trace amounts.⁶ The proenzyme is activated only after it reaches the lumen of the small intestine. Enterokinase activates pancreatic trypsinogen to trypsin by the hydrolysis of a hexapeptide(for bovine trypsin at the Lys⁶ - Ile⁷ peptide bond) from the NH₂ terminus. Bovine trypsinogen consists of a single polypeptide chain of 229 amino acids and is cross linked by six disulfide bridges. Trypsin can autocatalytically activate more trypsinogen to trypsin. Trypsin consists of a single chain polypeptide of 223 amino acid residues. This native form of trypsin is refered to as β-trypsin. Autolysis of β-trypsin (which is cleaved at Lys¹³¹- Ser¹³² in the bovine sequence) results in α-trypsin which is held together by disulfide bridges. Trypsin is a member of the serine protease S1 family. The active site amino acid residues of trypsin include His⁴⁶ and Ser¹⁸³.^2-5

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Specificity, Kinetics, Substrates and Assay Methods

Specificity and Kinetics
Trypsin will cleave peptides on the C-terminal side of lysine and arginine amino acid residues. The rate of hydrolysis is slower if an acidic residue is on either side of the cleavage site and no cleavage occurs if a proline residue is on the carboxyl side of the cleavage site.

Trypsin will hydrolyze ester and amide linkages of several synthetic substrates: ^2,7,8

Additional Substrates

The pH optimum of trypsin is 7 - 9.¹⁰

Assay Method¹¹
The activity of most Sigma preparations is determined by a continuous rate spectrophotometric assay and expressed in BAEE units.

Unit Definition: One BAEE unit will produce a ΔA₂₅₃ of 0.001 per min at pH 7.6 at 25 °C using BAEE as substrate. Reaction volume = 3.2 mL (1 cm light path).

Conditions: Temp = 25 °C, pH = 7.6, A₂₅₃nm, Light path = 1 cm
In a 3.2 ml reaction mix, the final concentrations are 63 mM sodium phosphate, 0.23 mM Nα-benzoyl-L-arginine ethyl ester, 0.06 mM hydrochloric acid, and 100 units trypsin.

Reagents:

1. 67 mM Sodium Phosphate Buffer, pH 7.6 at 25 °C (Prepare 100 ml in deionized water using Sodium Phosphate, Monobasic, Anhydrous, Sigma Prod. No. S0751. Adjust to pH 7.6 at 25 °C with 1 M NaOH.)
2. 0.25 mM Na-Benzoyl-L-Arginine Ethyl Ester Solution (BAEE) (Prepare 50 ml in Reagent A using Nα-Benzoyl-L-Arginine Ethyl Ester, Hydrochloride, Sigma Prod. No. B4500.)
3. 1 mM Hydrochloric Acid Solution (HCl) (Prepare 50 ml in deionized water using concentrated Hydrochloric Acid, Sigma Prod. No. 258148.)
4. Trypsin Enzyme Solution (Immediately before use, prepare a solution containing 500 BAEE units/ml of Trypsin in cold Reagent C.)

Procedure:

Pipette (in milliliters) the following reagents into suitable quartz cuvettes:

Equilibrate to 25 °C. Monitor the A_253nm until constant, using a suitably thermostatted spectrophotometer. Then add:

Immediately mix by inversion and record the increase in A_253nm for approximately 5 minutes. Obtain the ΔA_253nm/minute using the maximum linear rate for both the Test and Blank.

Calculation:

df = Dilution factor
0.001 = The change in A_253nm/minute per unit of Trypsin at pH 7.6 at 25 °C in a 3.2 ml reaction mix
0.20 = Volume (in milliliters) of enzyme used

Notes:

1. This assay procedure is not to be used to assay immobilized trypsins
2. For the USP/NF procedure refer to the USP monograph
3. This procedure is for informational purposes. For a current copy of Sigma’s quality control procedure contact our Technical Service Department.

Unit Conversions

1 BAEE µM Unit = 200 BAEE Units
1 TAME µM Unit = 0.27 BAEE µM Units
1 BAEE µM Unit = 3.64 TAME Units
1 TAME µM Unit = 55 BAEE A₂₅₃ Units
1 BAEE A253 Unit = 0.018 TAME µM Unit
1 TAME µM Unit = 180 TAME A₂₄₇ Units
1 TAME A₂₄₇ Unit = 0.33 BAEE Units
1 USP Unit = 3.0 BAEE Units
1 NF Unit = 1.1 USP Units

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Inhibitors

Serine protease inhibitors that will inhibit trypsin include: ^2,11

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Reconstitution and Solution Stability

Trypsin solutions in 1 mM HCl (pH 3) are stable for approximately 1 year when aliquoted and stored at -20 °C. The presence of Ca²⁺(20 mM) will also retard trypsin's ability to selfdigest itself (autolysis) and will maintain the stability of the trypsin in solution.^2,9 Trypsin retains most of its activity in 2.0 M urea, 2.0 M guanidine HCl, or 0.1% (w/v) SDS.¹³ Trypsin is reversibly denatured at high pH (above 11), by precipitation with TCA, or by high concentrations of urea (greater than 6.5 M).³ In order to abolish all trypsin activity, heating at 100 °C in 1% (w/v) SDS for 5 minutes is required.¹⁴
Trypsinogen solutions are stable in acidic buffers (pH 2 - 4), while in neutral buffers the autocatalytic activation to trypsin occurs.

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Applications and Protocols

Cell Dissociation
Mitochondria Isolation
Protein Sequencing

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Products

Trypsin for General Research Applications

Bovine Trypsin

Porcine Trypsin

Human Trypsin
Trypsin from human pancreas, salt-free, lyophilized powder, vial of ≥1,000 BAEE units

Trypzean™ Recombinant Bovine Trypsin, Expressed in Corn
TrypZean Powder
TrypZean Solution

Protein Sequencing
Trypsin Products for Proteomic/Protein Sequencing Analysis

Trypsin Solutions for Cell Dissociation

Trypsinogen

References

1. Cunningham, L. W. Jr., Molecular Kinetic properties of crystalline diisopropyl phosphoryl trypsin. J. Biol. Chem., 211, 13-19 (1954).
2. Walsh, K. A., Trypsinogens and trypsins of various species. Meth. Enzymol., 19, 41-63 (1970).
3. Keil, B., in The Enzymes, 3rd ed., Vol. III, Boyer, P. D., Academic Press (New York, NY: 1971), pp. 250-275.
4. Shaw, E., et al., Evidence for an active center histidine in trypsin through use of a specific reagent, 1-chloro-3-tosylamido-7-amino-2-heptanone, the chloromethyl ketone derived from Nα-tosyl-L-lysine. Biochemistry, 4(10), 2219-2224 (1965).
5. Tietze, F., Molecular kinetic properties of crystalline trypsinogen. J. Biol. Chem., 204(1), 1-11 (1953).
6. Chen, J-M; Ferec, C., Genes, Cloned cDNAs, and Proteins of Human Trypsinogens and Pancreatitis-Associated Cationic Trypsinogen Mutations. Pancreas, 21, 57-62 (2000)
7. Burdon, R. H., et al., in Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 9, 2nd ed., Allen, G., ed., Elsevier/North (New York, NY: 1989), pp. 73-104.
8. Enzyme Handbook, Vol. II, Barman, T. E., Springer-Verlag (New York, NY: 1969), pp. 618-619.
9. Enzyme Handbook, Vol. II, Barman, T. E., Springer-Verlag (New York, NY: 1969), p. 819.
10. Sipos, T., and Merkel, J. R., An effect of calcium ions on the activity, heat stability, and structure of trypsin. Biochemistry, 9(14), 2766-2775 (1970).
11. Bergmeyer, H.U., Gawehn, K., and Grassl, M. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed) Volume I, 2nd ed., 515-516, Academic Press, Inc.,New York, NY
12. Wang, S. S., and Carpenter, F. H., Kinetics of the tryptic hydrolysis of the oxidized B chain of bovine insulin. Biochemistry, 6(1), 215-224 (1967).
13. Carpenter, F. H., Treatment of trypsin with TPCK. Methods Enzymol., 11, 237 (1967).
14. Methods of Molecular Biology, Vol. 3, Smith, B. J., Humana Press, (New Jersey, 1988), pp. 57-69.

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