Enzyme Explorer

Trypsin

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Physical Properties and In Vivo Processing

Trypsin
Enzyme Commission (EC) Number: 3.4.21.4
Molecular Weight: 23.3 kDa1,2 (bovine & porcine)
Extinction Coefficient: E1% = 12.9 - 15.4 (280 nm)3,4
pI: 10.1 - 10.52,5(bovine)

Trypsinogen
Molecular Weight: 24 kDa5 (bovine)
Extinction Coefficient: E1% = 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


Product #  Product Name Add to Cart
B7260 Nα-Benzoyl-L-arginine-7-amido-4-methylcoumarin hydrochloride
B4500 Nα-Benzoyl-L-arginine ethyl ester hydrochloride
B0885 Nα-Benzoyl-L-arginine 4-methoxy-β-naphthylamide hydrochloride
B4750 Nα-Benzoyl-DL-arginine β-naphthylamide hydrochloride
B3133 Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride ≥99% (TLC), suitable for substrate for trypsin
B4875 Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride ≥98%

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:

  Test Blank
Reagent B (BAEE) 3.00 3.00

Equilibrate to 25 °C. Monitor the A253nm until constant, using a suitably thermostatted spectrophotometer. Then add:

  Test Blank
Reagent C (HCl) --- 0.20
Reagent D (Enzyme Solution) 0.20 ---

Immediately mix by inversion and record the increase in A253nm for approximately 5 minutes. Obtain the ΔA253nm/minute using the maximum linear rate for both the Test and Blank.

Calculation:


df = Dilution factor
0.001 = The change in A253nm/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


Product #  Product Name Add to Cart
A6664 4-Amidinophenylmethanesulfonyl fluoride hydrochloride
A8456 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride ~97%
A6191 Antipain dihydrochloride from microbial source
A2221 Antithrombin III from human plasma lyophilized powder, ≥95% (SDS-PAGE)
A9141 Antithrombin III from bovine plasma lyophilized powder, 200-400 units/mg protein (E1%/280 = 6.5)
A6150 α1-Antitrypsin from human plasma salt-free, lyophilized powder
A9024 α1-Antitrypsin from human plasma salt-free, lyophilized powder
A1153 Aprotinin from bovine lung lyophilized powder, 3-8 TIU/mg solid
B3906 Bdellin from leeches lyophilized powder, >0.1 IU/mg protein (E280)
B6506 Benzamidine hydrochloride hydrate ≥98%
D0879 Diisopropyl fluorophosphate
D7910 3,4-Dichloroisocoumarin
G2417 Gabexate mesylate solid
L2023 Leupeptin trifluoroacetate salt ≥90% (HPLC), microbial
M6159 α2-Macroglobulin from human plasma lyophilized powder, ≥98% (SDS-PAGE)
P7626 Phenylmethanesulfonyl fluoride ≥98.5% (GC)
T7254 Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride ≥96% (TLC), powder
T0256 Trypsin inhibitor from bovine pancreas Type I-P, essentially salt-free, lyophilized powder
T2011 Trypsin inhibitor from chicken egg white Type III-O (free of ovoinhibitor)
T4385 Trypsin inhibitor from turkey egg white Type II-T
T9003 Trypsin inhibitor from Glycine max (soybean) Type I-S, lyophilized powder
T9008 Trypsin inhibitor from Glycine max (soybean) 1% solution in water (contains no buffers)
T9253 Trypsin inhibitor from chicken egg white Type II-O, Partially purified ovomucoid, containing ovoinhibitor
T9378 Trypsin inhibitor from Phaseolus limensis (lima bean) Type II-L, crude powder

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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

Product #  Product Name Add to Cart
T1426 Trypsin from bovine pancreas essentially salt-free, lyophilized powder, ≥10,000 BAEE units/mg protein
T8003 Trypsin from bovine pancreas Type I, ~10,000 BAEE units/mg protein
T1005 Trypsin from bovine pancreas Type XI, lyophilized powder, ≥6,000 BAEE units/mg protein
T8658 Trypsin from bovine pancreas suitable for protein sequencing, lyophilized powder
T7309 Trypsin from bovine pancreas ≥2,500 USP units/mg solid, meets USP testing specifications
T9935 Trypsin from bovine pancreas essentially salt-free, lyophilized powder, 9,000-13,000 BAEE units/mg protein, cell culture tested
T8802 Trypsin from bovine pancreas essentially salt-free, lyophilized powder, 10,000-15,000 BAEE units/mg protein
T6424 Trypsin from human pancreas salt-free, lyophilized powder, vial of ≥1,000 BAEE units
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Porcine Trypsin

Product #  Product Name Add to Cart
T0303 Trypsin from porcine pancreas Type IX-S, lyophilized powder, 13,000-20,000 BAEE units/mg protein
T4799 Trypsin from porcine pancreas lyophilized powder, 1,000-1,500 BAEE units/mg solid, cell culture tested
T7409 Trypsin from porcine pancreas lyophilized powder, Type II-S, 1,000-2,000 BAEE units/mg solid
T5266 Trypsin from porcine pancreas 1,000-1,500 BAEE units/mg solid, γ-irradiated, cell culture tested
T7168 Trypsin from porcine pancreas tablet, 1 mg tablet
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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

Product #  Product Name Add to Cart
T4549 Trypsin solution from porcine pancreas 10 ×, 25 g porcine trypsin per liter in 0.9% sodium chloride, sterile-filtered, cell culture tested
T4674 Trypsin solution from porcine pancreas 10 ×, 25 g porcine trypsin per liter in Hanks' Balanced Salt Solution with phenol red, sterile-filtered, cell culture tested
T4424 Trypsin solution from porcine pancreas 1 ×, 2.5 g porcine trypsin per liter in Hanks' Balanced Salt Solution with phenol red, sterile-filtered, cell culture tested
T4049 Trypsin-EDTA solution 0.25%, 2.5 g porcine trypsin and 0.2 g EDTA • 4Na per liter of Hanks' Balanced Salt Solution with phenol red, sterile-filtered, cell culture tested
T3924 Trypsin-EDTA solution 1 ×, 0.5 g porcine trypsin and 0.2 g EDTA • 4Na per liter of Hanks' Balanced Salt Solution with phenol red., sterile-filtered, cell culture tested
T4299 Trypsin-EDTA solution 1 ×, 500 BAEE units porcine trypsin and 180 μg EDTA • 4Na per ml in Dulbecco's Phosphate Buffered Saline without calcium and magnesium., sterile-filtered, cell culture tested
T4174 Trypsin-EDTA solution 10 ×, 5.0 g porcine trypsin and 2 g EDTA • 4Na per liter of 0.9% sodium chloride, sterile-filtered, cell culture tested
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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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