Proteases and Protease Inhibitors

Simply put, proteases digest proteins and peptides. They are abundant in every organism inside their cells and here fulfill a crucial role in regulating the life cycle of proteins, activating pro-enzymes or eliminating problematic proteins. In addition, proteases are secreted to process food, e.g. in the intestinal tract of animals.

Protease Inhibitors used in protein purification procedures

Whenever proteins are analyzed in biological samples or purified from a natural source, protease activity is a potential threat. During sample preparation, cells are frequently lysed and in this way they set free high amounts of protease activities that may digest the proteins of interest. The days of work for cell culture and protein sample preparation can be destroyed within a few seconds.Labs generally apply two basic strategies to knock out such unwanted proteolytic activities: (a) cooling the sample or cell lysate, and (b) adding chemical inhibitors of proteases.
The most common proteases are the serine proteases Chymotrypsin, Kallikrein, Plasmin, Proteinase K, Thrombin and Trypsin. Hence their deactivation after cellular homogenization is very important in the isolation of proteins in order to ensure satisfactory protein purification yields.

Case Study: Using AEBSF, PMSF or DFP?

Phenylmethylsulfonyl fluoride, PMSF (P-4170) and 4-(2-Aminoethyl)-benzenesulfonylfluoride hydrochloride, AEBSF (A-5440) are irreversible serine protease inhibitors and are, thus, part of most homogenization buffers and added to cell lysates. Deactivation (irreversible inhibition) occurs through esterification of the serine hydroxyl group.
PMSF not only deactivates serine proteases but also any other enzyme that contains serine in its active site. Therefore PMSF cannot be used if the biological activity of such an enzyme needs to be maintained.
AEBSF is often preferred over PMSF or DFP (diisopropylfluorophosphate) because it is less toxic and water soluble. Aqueous AEBSF solutions are stable at slightly acidic pH values.

Benzamidine hydrochloride:
Benzamidine (B-0500, Q101108) is a competitive inhibitor of serine proteases. Benzamidine is frequently added to cell lysates, especially yeast cell extracts. It’s also a preferred protease inhibitor in protein crystallography.
p-Aminobenzamidine immobilized on agarose is used in the isolation of serine proteases by affinity chromatography.

Aprotinin from bovine lung:
Aprotinin is a small protein but a powerful inhibitor that prevents activity of several serine proteases (trypsin, chymotrypsin, plasmin, and kallikrein) already at low inhibitor concentrations.

Metalloproteases are also common. They are readily deactivated by the addition of EDTA. EDTA binds the metal ions that the enzymes need to function. Other proteases including aspartic acid and cysteine proteases are less important but may need to be deactivated with appropriate reagents nonetheless. Agents such as N-ethylmaleimide (NEM) (E-8100) or 4-Chloromercuribenzoic acid (C-5150) which modify thiol groups can be used as cysteine protease inhibitors.

Protease Inhibitors: Overview

InhibitorEnzymes
AEBSF (A-5540) Serine Proteases
6-Aminocaproic acid (A-5295) Serine Proteases 
Aprotinin (J-200086) Plasmin, Kallikrein, Trypsin, Chymotrypsin
Benzamidine (B-0500) Serine Proteases
E-64 Cysteine Proteases (Papain, Cathepsin)
EDTA (J-610078) Metalloproteases
EGTA (W-104715) Ca(II)-Proteases 
Leupeptin Serin and Cysteine Proteases 
Pepstatin Aspartic Proteases (eg. HIV)
PMSF (P-4170) Serine Proteases 



Please click here to see our list of commonly used protease inhbitors.

Example of a typical homogenization buffer

Tris-HCI (pH 7.6), 20 mmol/L
EDTA, 5 mmol/L
EGTA, 5 mmol/L
DTT, 1,4-Dithiothreitol (D-8200), 1 mmol/L
PMSF, Phenylmethylsulfonyl fluoride (P-4170), 0.5 mmol/L
AEBSF, 4-(2-Aminoethyl)-benzenesulfonylfluoride hydrochloride (A-5440), 10 mg/L
Leupeptin, 5 mg/L

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