Enzyme Explorer

Tryptase

Tryptase is a glycoprotein released from mast cells during anaphylaxis, which performs a number of functions including catalyzing the activation of complement C3,7 converting prostromelysin to stromelysin (MMP-3),8 and cleaving fibrinogen resulting in a loss of clotting potential.9


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Tryptase from Human Lung T7063


Physical and Physiological Properties

Tryptase
Enzyme Commission (EC) Number: 3.4.21.59

Molecular Weight: ~135 kDa (Human)
     Non-covalently linked tetramer with two sets of dissimilar
     subunits possibly resulting from heterogeneity in N-linked
     glycosylation and existance of α & β isoforms sequences
     in human lung1,2
  31-33 kDa3,4 (Human)
     Monomer MW

Tryptase is a tetrameric glycoprotein and a member of the serine protease S1 family. It is the predominant neutral protease of the mast cell granules. Within the mast cell granule it exists as a heparin-stabilized active tetramer. Stabilization is a result of the high negative charge density of the glycosaminoglycan. This stabilization activity is observed with heparins with a MW greater than 6 kDa as well as other glycosaminoglycans such as dextran sulfate, chondroitin sulfates.3 Removal of heparin results in dissociation of the tetramer and inactivation of the enzyme.3 High concentrations of NaCl will result in the dissociation of heparin.3,4

Tryptase is released from the mast cell as a result of the degranulation response during anaphylaxis.5 In addition, several tryptase genes and alleles (α,β,γ & δ) have been identified in various tissues and circulating in blood. Pro-β-tryptase is thought to be the constituative circulating form in blood.6

The biological function of tryptase is unknown. However it has been reported to catalyze the activation of complement C3,7 convert prostromelysin to stromelysin (MMP-3),8 and cleave fibrinogen resulting in a loss of clottting potential.9 Tryptase also degrades fibronectin,10 calcitonin gene-related peptide,11 vasoactive intestinal peptide,12 and kininogen. 13


Tryptase Domain Structure

Specificity, Kinetics, Inhibitors and Substrates


Specificity and Kinetics

Tryptase will cleave peptides on the C-terminal side of lysine and arginine amino acid residues with more strict specificity than trypsin.15


Substrates

N-Acetyl-Lys-Pro-Arg-7-amido-4-trifluoromethylcoumarin, C6608
Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride, B3133
Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride, B4875

The pH optimum for activity of tryptase is 8-9.13,14

Assay Method

The activity of tryptase is determined by a continuous rate spectrophotometric assay and expressed in BAPNA units.

Unit Definition: One unit will hydrolyze 1.0 micromole of N-benzoyl DL-arginine p-nitroanilide (BAPNA) per minute at pH 7.8 at 37°C.

Tryptase Assay Reaction

Inhibitors

Unlike trypsin, the serine protease inhibitor proteins of human plasma, such as α2-macroglobulin, as well as soybean trypsin inhibitor do not inhibit tryptase.4 5 mM Ca+2 can cause irreversible loss of activity.16

Antipain, A6191
Benzamidine Hydrochloride, B6506
Diisopropyl fluorophosphate, D0879
Gabexate mesylate, G2417
Leupeptin, L2023
Phenylmethylsulfonyl fluoride (PMSF), P7626
N-α-p-Tosyl-L-lysine chloromethyl ketone hydrochloride, T7254
Histamine, H7125

Solution Stability


Sigma’s T7063 tryptase is supplied as a solution in 1M NaCl, 0.05 mM heparin, 50 mM sodium acetate, pH 5.0, containing 0.01% sodium azide. Recommended long-term storage is –20 °C . It can be stored for a short term at 4 °C.


References

  1. Rest, R., Meth. Enzymol., 163, 309-327 (1988).
  2. Schwartz, L. B., Meth. Enzymol., 244, 88-100 (1994).
  3. Miller, Jeffrey, et al., J. Clin. Invest., 84, 1188-1195 (1985).
  4. Smith, T.J., et al., J. Biol. Chem., 259, 11046-11051 (1984).
  5. Schwartz, L. B., et al., N. Engl. J. Med., 316, 1622-1626 (1987).
  6. Caughey, G. H., J. Allergy, Clin. Immunol., 117, 1411-1414 (2006).
  7. Schwartz, L. B., et al., J. Immunol., 130, 1891-1895 (1983).
  8. Lees, M., et al., E. J. Biochem., 223, 171-177 (1994).
  9. Schwartz, L. B., et al., J. Immunol., 135, 2762-2767 (1985).
  10. Lohi, J., et al., J. Cell. Biochem., 50, 337 (1992).
  11. Schwartz, L. B., Monogr. Allergy, 27, 90 (1990).
  12. Caughey, G. H., et al., J. Pharmacol. Exp., Ther., 244, 133 (1988).
  13. Walls, A.F., et al., Biochem., Soc. Trans., 20, 260S (1992).
  14. Peng, Q., et al., Eur. J. Biochem., 270, 270-283 (2003).
  15. IUBMB Enzyme Nomenclature: http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/21/59.html
  16. Alter, S.C., and Schwartz, L.B., Biochim. Biophys. Acta, 991, 426-430 (1989).

 

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