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

Protein Structure
Protein Structure
Fragmentation of Protein Chains - Characteristics of High-Specificity Proteases

Among Enzymes of this type, Trypsin is the most important. V8 Protease from S. aureus is also widely used. This enzyme hydrolyzes peptide bonds at dicarboxylic acid residues, predominantly glutamic acid. Recently, a novel protease has been isolated from a Pseudomonas fragi mutant, which hydrolyzes the bond at the amino group of aspartic and Cysteine residues.

Trypsin is a Serine protease and one of the most accessible and highly specific enzymes. It is obtained from various sources. The Specificity of trypsin is not absolute; it hydrolyzes peptide bonds on the carboxyl side of Lysine and Arginine with all other protein Amino Acids except Pro. The presence of acidic residues near the attacked bond sharply decreases the Hydrolysis rate or halts it altogether. Positively charged groups also reduce the rate.

Cases of non-specific trypsin action have been described, where peptide bonds located near aromatic or hydrophobic amino acid residues were hydrolyzed, as well as the Cleavage of Arg-Pro bonds in the sequences Try-Arg-Pro-Ala and Ala-Arg-Pro-Ala.

Hydrolysis conditions. Trypsin exhibits maximal activity at pH 7–9 and is reversibly inactivated at pH < 4. It is prone to autolysis (autodigestion), so the enzyme is dissolved in 10 mM HCl and stored in a frozen state. Under these conditions, it remains stable for weeks. Trypsin hydrolysis is carried out in 0.1 M ammonium bicarbonate buffer at an Enzyme-to-substrate ratio of 1:50–100, T = 37∘C, for 1–4 hours. The enzyme is irreversibly inactivated (inhibited) by DFP and PMSF. Several specific inhibitors are known for trypsin, such as the soybean trypsin inhibitor.

Thrombin is an accessible and highly specific enzyme, but it is practically never used for Protein Cleavage. The reason is likely that thrombin preparations commercially available from various companies vary widely in purity and activity degree.

V8 Protease from Staphylococcus aureus. This serine protease is isolated from an S. aureus strain and mass-produced as a commercial preparation.

Specificity: it cleaves predominantly the peptide bond on the carboxyl side of glutamic acid with any other amino acid except Pro and Glu. Substrate specificity is influenced by the COMPOSITION OF THE buffer mixture. Instances of non-specific activity are known, primarily affecting the Ser-X bond.

Hydrolysis conditions: pH 3.5–9.5, with maximal activity observed at two pH values: 4.0 and 7.8. Buffer: 50 mM ammonium acetate. T = 37∘C, hydrolysis time up to 18 hours. Inhibited by DFP. The enzyme retains its activity in a 0.2% sodium dansyl (SDS) solution and 50% activity in 4 M urea, making it suitable for use under denaturing conditions.

Clostripain is a sulfhydryl protease isolated from Clostridium histolyticum. The enzyme is commercially available.

Specificity: it hydrolyzes peptide bonds on the carboxyl side of arginine with any other amino acid. Occasionally, it cleaves the Lys-X bond. This pathway of hydrolysis can be restricted or prevented by adjusting conditions. Hydrolysis conditions: activity maximum at pH 7.7 in the presence of sulfhydryl Reagents. Ca²⁺ ions promote hydrolysis, although their presence is not strictly required. Enzyme-to-substrate ratio: 1:50. Buffer: 100 mM NH₄HCO₃ in dithiothreitol (DTT): SHCH₂-CH(OH)-CH(OH)-CH₂SH. Temperature: 25∘C, duration: 4 hours.

Mouse Submandibular gland protease is a serine protease isolated from the submandibular gland of mice. It cleaves peptide bonds on the carboxyl side of arginine with all amino acids, except for Arg-Val and Arg-Rounds bonds. In terms of specificity, the enzyme is analogous to clostripain, yet it practically does not affect Lys-X bonds.

Hydrolysis conditions: buffer - 1% NH₄HCO₃, pH 8, T = 37∘C, duration up to 24 hours, enzyme-to-substrate ratio - 1:50. Hydrolysis is inhibited by acidification of the medium with Hydrochloric acid to pH 1.5–2.

Armillaria mellea protease is a Zn²⁺-containing metalloprotein. The enzyme has been isolated and obtained as a pure active preparation with a Molecular Weight of 14,000 and a pH optimum at 6.8. It is not yet commercially produced.

Specificity. It hydrolyzes peptide bonds on the amino side of lysine and aminoethylcysteine (Aec) of the X-Lys type, even if X = Pro. Peptide bonds containing acidic residues near Lys residues are resistant to the enzyme's action. It occasionally cleaves the Arg-X bond (on the carboxyl side of Arg). The degree of hydrolysis increases if X = Leu, Ile.

Hydrolysis conditions: 0.2 M N-ethylmorpholine acetate buffer, pH 8, enzyme-to-substrate ratio - 1:100, T = 35∘C, duration up to 22 hours, or 0.1 M NH₄HCO₃, ratio 1:500–1000, T = 37∘C.

Post-Proline-specific enzyme. Proline is a rare amino acid. Currently, only one post-proline-specific enzyme, isolated from lamb Kidney, is known and tested. Preliminary data indicate it is a serine protease.

Specificity. Studies on short Peptides (no more than 13 amino acids) have shown that the enzyme cleaves all Pro-X bonds except Pro-Pro. The rate of hydrolysis depends on X; it is maximal when X possesses hydrophobic properties and minimal when X is an acidic or basic amino acid.

The enzyme affects Ala-X bonds, but the rate of hydrolysis is 100–1000 times lower than that of Pro-X bonds.

Hydrolysis conditions: pH 7.5–8.0 (activity maximum), buffer: 100 mM NH₄HCO₃ supplemented with 1 mM each of DTT and EDTA, temperature: 22∘C, duration: 24 h.