[Skip to Content](https://www.sigmaaldrich.com#main-content) [](https://www.sigmaaldrich.com/US/en) Products Cart0 USEN Products [Login / Register](https://www.sigmaaldrich.com/oidc-sign-in) [Order Lookup](https://www.sigmaaldrich.com/US/en/order-lookup) [Quick Order](https://www.sigmaaldrich.com/US/en/quick-order) Cart0 [Home](https://www.sigmaaldrich.com/US/en)[Protein Labeling & Modification](https://www.sigmaaldrich.com/US/en/applications/protein-biology/protein-labeling-and-modification)Chymotrypsin # Chymotrypsin *E.C. 3.4.21.1* - [Products](https://www.sigmaaldrich.com#products) - [Physical Properties](https://www.sigmaaldrich.com#physical) - [Specificity and Kinetics](https://www.sigmaaldrich.com#specificity) - [Inhibitors](https://www.sigmaaldrich.com#inhibitors) - [Substrates](https://www.sigmaaldrich.com#substrates) - [Solubility and Solution Stability](https://www.sigmaaldrich.com#solubility) ## [](https://www.sigmaaldrich.com)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  __Figure 1.__Physical Properties ## [](https://www.sigmaaldrich.com)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 [](https://www.sigmaaldrich.com) ## Inhibitors Sorry, an unexpected error has occurred Response not successful: Received status code 500 α-Chymotrypsin is also completely inhibited by 10 mM Cu2+ and Hg2+.1 [](https://www.sigmaaldrich.com) ## Substrates Sorry, an unexpected error has occurred Response not successful: Received status code 500 [](https://www.sigmaaldrich.com) ## 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. ## 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 [](https://www.sigmaaldrich.com) ## Products Sorry, an unexpected error has occurred Response not successful: Received status code 500 ### References 1\. Burrell MM. Enzymes of Molecular Biology. [https://doi.org/10.1385/0896032345](https://doi.org/10.1385/0896032345) 2\. Ui N. 1971. Isoelectric points and conformation of proteins. Biochimica et Biophysica Acta (BBA) - Protein Structure. 229(3):582-589. [https://doi.org/10.1016/0005-2795(71)90273-x](https://doi.org/10.1016/0005-2795%2871%2990273-x) 3\. Desnuelle P, Boyer P. 1960. The Structure of Chymotrypsin. The Enzymes. 3185-191. 4\. HESS G. 1971. The Enzymes. 3. New York: Academic Press. 5\. Burrell MM. Enzymes of Molecular Biology. [https://doi.org/10.1385/0896032345](https://doi.org/10.1385/0896032345) 6\. Spackman D, Stein W, Moore S. 1960. The disulfide bonds of ribonuclease.. Journal of Biological Chemistry. 3(255):648-659. 7\. Kamp RM. 1986. Separation of Peptides.8-20. [https://doi.org/10.1007/978-3-642-71534-1\_2](https://doi.org/10.1007/978-3-642-71534-1_2) 8\. Ásgeirsson B, Bjarnason JB. 1991. Structural and kinetic properties of chymotrypsin from atlantic cod (Gadus morhua). Comparison with bovine chymotrypsin. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry. 99(2):327-335. [https://doi.org/10.1016/0305-0491(91)90050-n](https://doi.org/10.1016/0305-0491%2891%2990050-n) 9\. Fernández M, Villalonga MdL, Fragoso A, Cao R, Villalonga R. 2002. Stabilization of ?-chymotrypsin by modification with ?-cyclodextrin derivatives. Biotechnol. Appl. Biochem.. 36(3):235. 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