Practical Protein Chemistry - A. Darbre 1989

Enzymatic fragmentation of the polypeptide chain
High-specificity proteases
Trypsin

One of the most accessible and widely used highly specific Enzymes is trypsin. Trypsin preparations are obtained from various sources, although they are sometimes contaminated with Chymotrypsin. In such cases, traces of chymotrypsin are inhibited using L-(1-tosylamido-2-phenylethyl) chloromethyl ketone (TPCK) [57]. It is recommended to use TPCK-treated trypsin supplied by Worthington.

3.5.1.1. Specificity and Hydrolysis conditions.

Specificity. Trypsin specifically cleaves peptide bonds at the carboxyl group of Lysine and Arginine, i.e., -Lys-X- and -Arg-X- type bonds. However, the enzyme's specificity is not absolute; for instance, the -Lys-Pro- and -Arg-Pro- sequences are resistant to trypsin action. The presence of acidic residues near the bond being attacked leads to a sharp decrease in The rate of hydrolysis, and in some cases completely prevents it. Positively charged groups also reduce the rate of hydrolysis. For example, if Arg and Lys are in close proximity or located at the N-terminus of the polypeptide chain, only partial Cleavage of the peptide bonds occurs.

The literature provides A number of Examples of nonspecific trypsin action, although such instances are extremely rare. Peptide bonds located near aromatic or hydrophobic amino acid residues can be susceptible to the enzyme. It has been shown that this type of hydrolysis is not associated with chymotrypsin contamination, but is a property of trypsin itself or of ψ-trypsin present as an impurity [52]. In some cases, the cleavage of -Arg-Pro- bonds is observed, for example in the -Trp-Arg-Pro-Ala- [74] and -Ala-Arg-Pro-Ala- [35] fragments. The number of resulting Peptides may not correspond to the total amount of arginine and lysine residues (plus one). Peptides containing a lysine or arginine residue not located at the C-terminal region of the peptide can be obtained, though such cases are exceedingly rare. Therefore, hasty Conclusions should be avoided when interpreting the obtained results.

Hydrolysis conditions. Trypsin is a Serine protease exhibiting maximum activity in the pH range of 7–9. The enzyme is reversibly inactivated at pH < 4. To prevent autolysis, trypsin is dissolved in 10 mM HCl, after which the sample can be stored frozen for several weeks. Trypsin hydrolysis is performed in 0.1 M ammonium bicarbonate buffer at an Enzyme-to-substrate ratio of 1:50–100 at 37 °C for 1–4 hours. Hydrolysis can be limited by using a native protein as the substrate or by lowering the Temperature and incubation time. For example, at 25 °C for 30 minutes, cleavage of only 6 out of 15 trypsin-sensitive bonds is observed in troponin C [58].

Trypsin activity drops under denaturing conditions; for example, only 48% of activity is retained in the presence of 4 M urea [39]. Satisfactory results have been obtained for trypsin hydrolysis in 2 M guanidine-HCl [101]. The enzyme is irreversibly inactivated in the presence of DFP and PMSF. A number of specific inhibitors are also known, such as the soybean inhibitor [4], which forms a stoichiometric complex with trypsin and is often used to stop the reaction [58]. In this case, it is recommended to add 4 mg of the inhibitor per 1 mg of the enzyme to the reaction mixture. Hydrolysis can be stopped by acidification; however, the inactivation is reversible, and the enzyme activity is restored upon increasing the pH.

Specific Cleavage at arginine residues. Trypsin specificity can be restricted through the reversible blocking of lysine residues (Section 3.4.3). In this case, hydrolysis proceeds exclusively at arginine residues. Citraconylation is the most common modification; if the duration of trypsin hydrolysis is too long, maleylation is used. Deblocking of the maleyl or citraconyl groups is carried out by acidifying the reaction mixture to pH < 3.5, followed by the Separation of the resulting peptides. Upon acidification, the enzyme is inactivated; however, to prevent its reactivation during a subsequent pH increase, it is recommended to separate it from the reaction products or inactivate it irreversibly. If the modified peptides are to be handled in an acidic environment, introducing trifluoroacetyl groups into the peptide is recommended.

Specific cleavage at lysine residues. When Protein Hydrolysis is required solely at lysine residues, reversible Modification of arginine residues is employed [75] (Section 3.4.4). The resulting peptides are fractionated at low pH, and the protective groups are subsequently removed. Reproducible results are obtained using this method [5, 122]; however, in some cases, a decrease in Protein solubility is observed after modification, particularly following lyophilization.

Specific Cleavage at Cysteine residues. Alkylation of cysteine residues with ethylenimine yields a lysine analogue, S-aminoethylcysteine [81], thereby creating additional sites sensitive to trypsin action. The rate of hydrolysis at aminoethylcysteine residues is significantly lower compared to that at lysine and arginine residues [12, 78]. Provided that lysine and arginine residues are modified, trypsin hydrolysis proceeds selectively at cysteine residues. In this case, the Modification of lysine and arginine residues must precede reduction and subsequent aminoethylation. Conversely, in the case of citraconylation, The sulfhydryl groups of the protein are modified beforehand. The acidic Nature of the lysine protecting groups and the bulky side chains of the modified arginine residues prevent trypsin cleavage if one of these residues is located close to an aminoethylcysteine residue.

Specific cleavage at aspartic acid residues. The method of modifying carboxyl groups with diamines [112] allows for the selective Hydrolysis of Proteins at aspartic acid residues. This process involves the Modification of the C-terminal and glutamic acid carboxyl groups. Consequently, it is unlikely that this method will find Structure/182.html">Practical Application for protein hydrolysis. However, the method is quite suitable for the hydrolysis of short peptides containing a limited amount of glutamic acid or none at all.