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Merck
CN

Chymotrypsin

E.C. 3.4.21.1

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

Physical Properties

Figure 1.Physical Properties

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

Inhibitors
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α-Chymotrypsin is also completely inhibited by 10 mM Cu2+ and Hg2+.1

Substrates
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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.

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

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References

1.
Burrell MM. Enzymes of Molecular Biology. 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
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
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
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
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. https://doi.org/10.1042/ba20020056
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