Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022
Protein Structure
Protein Structure
Fragmentation of Protein Chains - Enzymatic Fragmentation
Mammalian digestive Enzymes are most frequently used for the Enzymatic Cleavage of Proteins into fragments. Examples of such enzymes include Trypsin, Chymotrypsin, and several others. Unlike aminopeptidases and Carboxypeptidases, which act on the terminal Amino Acids of peptide chains and are termed exopeptidases, trypsin, chymotrypsin, and similar enzymes cleave peptide bonds within the internal Links of the chain. Consequently, they are referred to as Endopeptidases.
All of these enzymes do not exhibit absolute Specificity toward The Nature of the bond being cleaved, yet under carefully optimized conditions, they still preferentially cleave specific bonds—meaning they exhibit so-called
"site specificity". Data on the specific Hydrolysis of peptide chains by certain enzymes are presented in Table 2.1.
Class="center">Table 2.1 Specific activity of certain enzymes in the cleavage of peptide chains.
Enzyme |
pH |
Main type of hydrolysis |
Secondary type of hydrolysis |
Unaffected bonds |
Trypsin |
7-9 |
Лиз-↓-Х, Арг-↓-Х, |
Лиз-Про |
|
8 |
Арг-↓-Х (Х=Гли, Ала, Вал, Цис, Арг, Асп |
|||
Protease V8 from Staphylococus aureus (S.aureus) |
4 and 7.8 |
Глу-↓-Х |
Асп-↓-Х |
Глу-Про Гли-Глу |
Clostripain |
7.7 |
Арг-↓-Х |
Лиз-↓-Х |
|
Protease from mouse submandibular gland |
7.5 8 |
Арг-↓-Х |
Арг-Вал Арг-Арг |
|
Protease from A.mellea |
8 |
Лиз-↓-Х |
Арг-↓-Х |
Х-Про |
Myxobacter AL1 protease |
9 |
Х-↓-лиз |
||
Postproline-specific protease |
7.5 8 |
Про-↓-Х |
Про-Про |
|
|
Proteases with broad specificity |
||||
Chymotrypsin |
7-9 |
Н-↓-Х (Н=Тир,Фен,Три,Лей) |
Н-Про |
|
7-8 |
Х-↓-Н (Н=Вал,Лей,Иле,Фен,Тир,Три) |
Х↓*Н-Про |
||
α-Protease from Crotalus Atrox |
7.5-8 |
Х4-Н (Н=Вал,Лей,Иле,Фен,Тир,Три) |
||
2 |
Х↓Н↓Y, X↓Глу↓Y (H=ароматический или объемный алифатический остаток) |
|||
5 7.5 |
Фен-Х↓Y. Идет Гидролиз и некоторых других пептидных связей. |
|||
7-9 |
N-↓-Y (N=небольшой нейтральный или гидрофобный остаток Сер,Ала,Гли,Вал,Лей) |
|||
α-Lytic protease |
7-8 |
N-↓-Y (Аналогично эластазе) |
||
Selecting optimal conditions for Enzymatic hydrolysis generally comes down to optimizing:
> buffer composition;
> Temperature;
> reaction time.
Buffer systems. Easily volatile buffer mixtures are typically used, such as 1% ammonium bicarbonate and 100 mM N-ethylmorpholine acetate.
Enzyme-to-substrate ratio - maintained at 1:50-100.
Temperature - in most cases, 20–37 °C. Only two enzymes, thermolysin and the protease from myxobacteria, retain high activity at temperatures > 40 °C.
Duration of hydrolysis - typically 2–4 hours. For refractory bonds that are difficult to cleave, the time is extended to 16–24 h.
The enzymatic hydrolysis reaction can be terminated after a specified time interval by means of:
> freezing;
> changing the pH of the medium;
> adding specific inhibitors.
Proteolytic Enzymes are divided into three main classes:
> Serine proteases;
> sulfhydryl proteases;
> metalloenzymes.
Specific inhibitors of serine proteases include diisopropyl fluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF).
The latter is preferred because DFP is highly toxic.
Sulfhydryl proteases are inhibited by an excess of alkylating agents, such as iodoacetic acid. Metalloenzymes are inhibited by chelating agents, notably ethylenediaminetetraacetic acid (EDTA).
Last update: 06/08/2026
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