Enzyme Explorer

Chymotrypsin

E.C. 3.4.21.1

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Physical Properties
Molecular Weight: 25 kDa1 (Bovine)
pI: 8.752 (Bovine)
Extinction coefficient: E1%= 20.4 (280nm)

Chymotrypsin is produced in the acinar cells of the pancreas as the inactive precursor, chymotrypsinogen. α-Chymotrypsin is the predominant form of active enzyme produced from it's zymogen, Chymotrypsinogen A.

In vivo, the rate of hydrolysis of the zymogen by trypsin and by autolysis produces varying amounts of α, π, δ and γ variants.3 α-Chymotrypsin is a serine protease of the peptidase S1 family consisting of 241 amino acid residues. The molecule has three peptide chains: an A chain of 13 residues, a B chain of 131 residues, and a C chain of 97 residues.4

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Specificity and Kinetics
α-Chymotrypsin from bovine pancreas selectively catalyzes the hydrolysis of peptide bonds on the C-terminal side of tyrosine, phenylalanine, tryptophan, and leucine. A secondary hydrolysis will also occur on the C-terminal side of methionine, isoleucine, serine, threonine, valine, histidine, glycine, and alanine.1

pH optimum: 7.88
(pH 6.0: about 35% of maximal activity, pH 9.3: 40% of maximal activity)8

Temperature Optimum: 50 °C9

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Inhibitors
Product #  Product Name Add to Cart
A8849 α2-Antiplasmin from human plasma lyophilized powder, ≥5 inhibitor U/mg protein
A1153 Aprotinin from bovine lung lyophilized powder, 3-8 TIU/mg solid
A2504 6-Aminocaproic acid ≥99% (titration), powder
A6150 α1-Antitrypsin from human plasma salt-free, lyophilized powder
A8456 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride ~97%
A9024 α1-Antitrypsin from human plasma salt-free, lyophilized powder
A9285 α1-Antichymotrypsin from human plasma ~95% (SDS-PAGE), lyophilized powder
B1552 Bromoenol lactone ≥98%
C7268 Chymostatin microbial
D0879 Diisopropyl fluorophosphate
E7888 N-Acetyl-eglin C recombinant, expressed in proprietary host
G2417 Gabexate mesylate solid
L2023 Leupeptin trifluoroacetate salt ≥90% (HPLC), microbial
T0256 Trypsin inhibitor from bovine pancreas Type I-P, essentially salt-free, lyophilized powder
T1886 Trypsin inhibitor from chicken egg white Type IV-O, Purified Ovoinhibitor
T4376 N-p-Tosyl-L-phenylalanine chloromethyl ketone ≥97% (TLC), powder
T4385 Trypsin inhibitor from turkey egg white Type II-T
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
T9777 Trypsin-chymotrypsin inhibitor from Glycine max (soybean) lyophilized powder
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α-Chymotrypsin is also completely inhibited by 10 mM Cu2+ and Hg2+.1


Substrates
Product #  Product Name Add to Cart
S7388 N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide
S9761 N-Succinyl-Ala-Ala-Pro-Phe-7-amido-4-methylcoumarin
1530 N-Acetyl-L-tyrosine ethyl ester monohydrate BioChemika, ≥99.0% (sum of enantiomers, HPLC)
A3401 Ala-Ala-Phe-7-amido-4-methylcoumarin
A9273 Ala-Ala-Val-Ala p-nitroanilide
13110 Magnesium according to Grignard, ≥99.5%, turnings
B6125 N-Benzoyl-L-tyrosine ethyl ester
B6760 N-Benzoyl-L-tyrosine p-nitroanilide
13135 N-Benzoyl-L-tyrosine p-amidobenzoic acid sodium salt BioChemika, lyophilized, ≥98.0% (sum of enantiomers, HPLC)
G3769 Glutaryl-Ala-Ala-Phe-4-methoxy-β-naphthylamide
49737 Glutaryl-L-phenylalanine 7-amido-4-methylcoumarin BioChemika, for fluorescence, ≥98.0% (HPLC)
49738 N-Glutaryl-L-phenylalanine p-nitroanilide BioChemika, ≥98.0% (HPLC)
49742 N-(N-Glutaryl-L-phenylalanyl)-2-aminoacridone BioChemika, for fluorescence, ≥98.0% (HPLC)
M4507 4-Methylumbelliferyl p-trimethylammoniocinnamate chloride powder
85977 N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide BioChemika, ≥99.0% (HPLC)
S1899 N-Succinyl-Gly-Gly-Phe-p-nitroanilide
S2628 N-Succinyl-L-phenylalanine-p-nitroanilide
85992 N-(N-Succinyl-L-phenylalanyl)-2-aminoacridone BioChemika, for fluorescence, ≥98.0% (HPCE)
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Solubility and Solution Stability
Chymotrypsin is typically soluble in 1 mM HCl (2 mg/ml), yielding a clear solution.

Reconstitute in 1 mM HCl containing 2 mM CaCl2, aliquot, and store at -20 °C. Autolysis will occur when stored at a higher pH. The presence of calcium is also a stabilizer.5 Frozen aliquots are stable for approximately 1 week. Chymotrypsin is both activated and stabilized by Ca2+. The enzyme is active in the presence of 0.1% SDS and 2 M guanidine hydrochloride.

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Applications
For peptide digestion, use a ratio (w/w) of approximately 1:60 for chymotrypsin:peptide. Perform peptide digests in 100 mM Tris HCl containing 10 mM CaCl2, pH 7.8, at 30 °C. Self digestion may occur if temperatures above 37 °C are used. A known peptide such as melittin should be used as a control for all experiments. Incubate up to 24 hours at 37-40 °C. Digestion can be terminated by adjusting the pH to 2.0.6,7


Products
Product #  Product Name Add to Cart
C4129 α-Chymotrypsin from bovine pancreas Type II, essentially salt-free, lyophilized powder, ≥40 units/mg protein
C7762 α-Chymotrypsin from bovine pancreas Type I-S, essentially salt-free, lyophilized powder
C6423 α-Chymotrypsin from bovine pancreas suitable for protein sequencing, salt-free, lyophilized powder
C3142 α-Chymotrypsin TLCK Treated from bovine pancreas Type VII, essentially salt-free, lyophilized powder, ≥40 units/mg protein
C9134 α-Chymotrypsin−Agarose from bovine pancreas lyophilized powder, 2,000-3,500 units/g agarose (One ml gel will yield 65-120 units)
C5088 α-Chymotrypsin−polyethylene glycol ~20 BTEE units/mg protein
C4879 α-Chymotrypsinogen A from bovine pancreas Type II, essentially salt-free, lyophilized powder, ≥40 units/mg solid (after activation to α-chymotrypsin.)
C1012 α-Chymotrypsin bovine Type VI (Inactivated with DFP), lyophilized powder, <0.2 units/mg protein
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Applications

  1. Enzymes of Molecular Biology, vol. 16, Burrell, M. M., ed., Humana Press (Totowa, NJ: 1993), pp. 277-281.
  2. Ui, N., Isoelectric points and conformation of proteins. II. Isoelectric focusing of alpha-chymotrypsin and its inactive derivative. Biochim. Biophys. Acta, 229(3), 582-589 (1971).
  3. Desnuelle, P.: The Structure of Chymotrypsin,The Enzymes, 3rd Ed. Vol. 3, P. Boyer, H. et al., Ed., Academic Press, NY, 185-191, 1960
  4. Hess, G. P., The Enzymes, 3rd ed., vol. 3, Boyer, P. D., ed., Academic Press (New York, NY: 1971), pp. 213-248.
  5. Burrell, M.M., Enzymes of Molecular Biology, Vol. 16, 278-281 (1993).
  6. Spackman, D.H., et al., J. Biol. Chem., 235, 648-659 (1960)
  7. Kamp, R. M. (1986) Advanced Methods in Protein Microsequence Analysis (Wittmann-Liebold, B., Salnikow, J., and Erdmann, V., eds) pp 8-20, Springer Verlag, Berlin
  8.  
  9. Asgeirsson, B.; Bjarnason, J.B.; Comp. Biochem. Physiol. B 99B, 327-335 (1991)
  10. Fernandez, M.; Villalonga Mde, L.; Fragoso, A.; Cao, R.; Villalonga, R.; Biotechnol. Appl. Biochem. 36, 235-239 (2002)

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