Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Protein Structure
Protein Structure
Protein Chain Fragmentation - Characteristics of Proteases with Broad Specificity

Below is a brief description of not all proteases in this Class, but rather the majority of those frequently used in laboratory practice.

Chymotrypsin is a Serine protease and one of the most specific Enzymes in this class. It is widely used to obtain primary hydrolysates.

Specificity: primarily hydrolyzes peptide bonds at the carboxyl side of aromatic or bulky hydrophobic amino acid residues of the type H-X, where X = Tyr, Trp, Phe, Leu. The H-Pro bond is resistant. It can also cleave at His, Ile, Met, Ser, and Val residues.

Hydrolysis conditions: 100 mM NH₄HCO₃, Enzyme-to-substrate ratio of 1 : 50, T = 37⁰C, time 4 h. Inhibited by soybean Trypsin inhibitor.

Thermolysin is widely used for protein amino acid sequencing and is commercially available as a prepared reagent.

Specificity: hydrolyzes peptide bonds at the amino side of hydrophobic amino acid residues of the type X-H, where, in most cases, H = Val, Leu, Ile, Phe, Tyr, Trp. It also readily hydrolyzes peptide bonds involving Ala and the Pro-Ile fragment. Hydrolysis at Asn, Thr, His, and Glu residues is rarely observed. Thermolysin hydrolysis is inhibited by adjacent α-amino or α-carboxylic groups; therefore, it exhibits no exopeptidase activity (toward peptide bonds with the terminal amino acid). Amino and carboxyl groups in the side chains of Amino Acids do not significantly affect enzyme activity.

The protease was named "Thermolysin" because it retains 100% activity for 1 h at T = 60°C and significant activity at 80°C. In addition, this enzyme is stable in 8 M urea, 20% ethanol, or methanol. Under these conditions, however, the probability of its thermal Denaturation increases.

Hydrolysis conditions: pH = 7 - 8, requires Ca²⁺ ions, buffer: 100 mM NH₄HCO₃ containing 5 mM CaCl₂, enzyme-to-substrate ratio 1 : 50, T = 37°C, time 4 h. The enzyme is inhibited by EDTA.

Pepsin is practically the only well-studied and commercially available acidic protease.

Specificity: predominantly cleaves peptide bonds involving aromatic and bulky aliphatic amino acid residues. Unfortunately, it is characterized by pronounced specificity toward A number of other peptide bonds as well, making the final outcome of partial hydrolysis difficult to predict. The enzyme is used for the Limited proteolysis of native Proteins.

Hydrolysis conditions: pH = 1 - 5 (pH optimum = 2), buffer: 10 mM HCl or 5% CH₃COOH. Incubation with pepsin yields short Peptides with correctly closed Disulfide Bonds. The enzyme is irreversibly inhibited at pH > 6.

Papain is a sulfhydryl protease isolated from the latex of the melon tree (papaya). Commercial preparations are supplied by several companies.

Specificity: in general, papain hydrolyzes A wide variety of peptide bonds, rendering the outcome of hydrolysis practically unpredictable. The enzyme has been used to obtain large fragments of native proteins, such as IMMUNOGLOBULINS, Myosin, the extracellular fragment of human Histocompatibility Antigens, etc.

Hydrolysis conditions: maximum activity in the pH range of 5 - 7.5; enzyme activation is carried out in the presence of sulfhydryl Reagents. Buffer: 0.2 M pyridine-acetate buffer (pH = 6.5) supplemented with 1% 2,3-dimercaptopropanol-1; enzyme-to-substrate ratio 1 : 50; T = 37°C; hydrolysis time 1 h. The enzyme is inactivated by oxidation with heavy metal salts or cyanate ions in the presence of Cysteine. It is stable in deionized urea solutions (up to 8 M urea).

Elastase is a serine protease homologous to trypsin and chymotrypsin, and is manufactured by several companies.

Specificity: exhibits broad specificity. It predominantly hydrolyzes peptide bonds at the carboxyl group of amino acids with a small hydrocarbon (hydrophobic) side-chain radical, such as Ala. Hydrolysis conditions are the same as for trypsin and chymotrypsin.

α-Protease from Crotalus atrox is isolated from venom and available commercially. Specificity: hydrolyzes peptide bonds at the amino side of hydrophobic amino acid residues. Its specificity is analogous to that of thermolysin. Hydrolysis conditions have not been definitively established. Maximum activity is in the pH range of 7.5 - 8.

In practice, trypsin, chymotrypsin, thermolysin, and proteinase V8 from Staphylococcus aureus are most frequently used.

A distinction is made between exhaustive and limited proteolysis. Exhaustive hydrolysis is typically applied to proteins in a denatured state, whereas native proteins are hydrolyzed along the globule surface. Limited proteolysis can be achieved under conditions where enzyme activity is reduced: lowered Temperature, pH values outside the optimal range, a decreased enzyme-to-substrate ratio, etc.