AppliCationsProteases are key enzymes in the regulation of cellular processes, they are found
everywhere in all cells and tissues. Upon cell lysis proteases are released into the
lysate. Some of the proteases pose a significant impediment to the analysis of
biochemical processes. They can generate erroneous results concerning the acti-
vity, structure, or location of proteins. Within only a few minutes protease activity
can destroy the preparations that took several days of work. Protease inhibitors are
employed in order to prevent involuntary protein degradation. They may be synthe-
sized in the laboratory or purified from natural sources.
Proteases (also termed proteinase or peptidase) catalyze the hydrolysis of peptide bonds. Exopeptidases remove amino acids from the C- or N-terminus, whereas endopeptidases are capable of cleaving peptides within the molecule. Proteolytic enzyme activity largely depends on the active center of the enzyme. The main components involved in the enzymatic reaction are the amino acids serine, cystein, and aspartic acid. A fourth group employs metal ions, leading to the classification of the metallo, serine, cystein, and aspartic proteases. In all eukaryotic cells and bacteria a large number of proteases are located in various compartments, the cytosol, mitochondria, vacuoles, lysosomes, ER, or in the extracellular space. Intracellular proteases are essential regulators in the synthesis, activation and degradation of proteins. Extracellular or secreted proteases are most prominent in the intestinal tract of animals or as a part of the blood-clotting cascade. Accordingly, different tissues or organisms contain different sets of proteases. Knowledge of the protease set of a particular expression system enables researchers to combat protease activity throughout the procedure of purification and analysis of proteins. As proteases evolved, specific natural inhibitors co-evolved, targeting the active center of the enzymes. Protease inhibitors are common in nature, where they have protective and regulatory functions. For instance, about 20 of the nearly 200 proteins of blood serum are protease inhibitors. Various mechanisms are characterized including chemical modification of proteases, competitive binding to the active site or competitive binding to cofactors. For example: (i) TLCK irreversibly inhibits trypsin by alkylating the histidine residue in the active site of the enzyme, (ii) Trypsin inhibitor from soybean forms a strong protein-protein interaction to the active site of trypsin (Fig. 1) and related serine proteases, (iii) α2-Macroglobulin traps endopepti-dases inside of the inhibitor, (iv) bestatine resembles a Phe-Leu substrate dipeptide, but the first residue contains a α-hydroxy group resulting in competitive active site-directed inhibition. Protease inhibitors bind to their target proteins reversibly or irreversibly. Reversibly binding inhibitors may be lost during dialysis or other purification steps. So it is of practical importance to know the mode of action for selecting the appropriate protease inhibitors and preparing solutions and buffers. Protease inhibitors are supposed to provide specificity so that proteases are blocked but other proteins stay unaffected. Other desired characteristics include solubility and stability. Ideally, the substances are also non-toxic and easy to handle. Scientists at research institutions have relied on the quality of AppliChem’s protease inhibitors for many years. AppliChem protease inhibitors are available as individual substances to target specific proteases (Tab. 1) or more convenient, as cocktails specifically designed to inhibit proteases of the most common expression systems (Tab. 2).
Keywords
Protein isolation
Protease activity
competitive inhibition
specific protease inhibitor
No.14Protease Inhibitors
Fig.1 Complex between trypsin inhibitor from soybean (a Kunitz-type proteina-se inhibitor) with its cognate proteinase. Trypsin inhibitor (above) selectively binds to the active site of the target protease (below) acting as a reversible competitive inhibitor (according to Song and Suh 1998).
Tab. 1: Individual Protease Inhibitors
Prod.-No.
Description M [g/mol] Structure Target Protease Class,Target Enzymes
Mechanism Recommen-ded Working Concentration
Stock Solution
A1421 AEBSF hydrochloride 239.69 serine proteases, thrombin, chymotrypsin, kallikrein, plasmin, proteinase K, trypsin
irreversibe inhibition by sulfonylation of a functional group in the active center
0.1–2 mM 20–100 mM in buffer
A3764 Amastatin 474.56 amino peptidases, aminopeptidase A, leucine aminopeptidase, many membrane-bound peptidases
competitive inhibitor of the aminopeptidases A and M (but not B)
1–10 µM 1 mM in water, MeOH, or EtOH
A2126 p-Aminobenzamidine dihydrochloride
208.09 serine proteases, trypsin, plasmin, thrombin
competitive inhibitor
1 mM 100 mM in water
A2266 ε-Aminocaproic acid 131.18 serine proteases 5 mM 500 mM in buffer
A2129 Antipain dihydrochloride
677.63 serine/cysteine proteases, trypsin, papain, cathepsin B
10–50 µg/ml 10 mg/ml in water, DMSO, MeOH
A0998 APMSF hydrochloride 252.69 serine proteases, trypsin, thrombin, factor Xa, plasmin
irreversible inhibitor of plasma serine proteases (trypsin-like proteases)
10–50 µM 1–5 mM in water
A2132 Aprotinin 6511.52 basic protein, consists of 58 amino acids
serine proteases, trypsin, chymotrypsin, kallikrein, plasmin
2–10 µg/ml 10 mg/ml in water
A1380 Benzamidine hydrochloride
156.62 serine proteases, trypsin, thrombin, plasmin
competitive inhibitor
1–5 mM up to 1 M in water
A2137 Bestatin hydrochloride
344.84 aminopeptidases, amino-peptidase B, leucine amino-peptidase, tripeptide amino-peptidase, aminopeptidases of the cell surface
competitive inhibitor
40 µg/ml (approx. 130 µM)
1–5 mg/ml in MeOH
A2144 Chymostatin 607.70 serine/cysteine proteases.α-, β-, γ-, δ-chymotrypsin, papain, cathepsin A, B and D
reversible inhibitor
6–60 µg/ml(10–100 µM)
20 mg/ml in DMSO, acetic acid
A2157 E-64 357.40 cysteine proteases, papain, bromelain, calpain, cathepsin B, H, L, tumor cathepsin, Streptococcus protease, ficin
irreversible inhibitor
10 µM 1–10 mM in DMSO, 50 % EtOH
A1103 EDTA 292.25 metallo proteases chelating agent, deactivates metal dependent enzymes
1–10 mM 500 mM in water
A0878 EGTA 380.35 metallo proteases, KEX 2, calcium-dependent proteases
calcium specific chelator
1–10 mM in aqueous solution
A1666 Iodoacetamide* 184.96 serine proteases, cerastocytin
1–5 mM(185–925 µg/ml)
in aqueous solution
A2097 Iodoacetic acid* sodium salt
207.93 serine proteases 1–5 mM(208–1040 µg/ml)
in aqueous solution
A2183 Leupeptin hemisulfate 475.60 serine/cysteine proteases, plasmin, trypsin, papain, cathepsin B, thrombin, calpain
reversible inhibitor 5–50 µg/ml(10–100 µM)
1 mg/ml in aqueous solutions
A2186 α 2-Macroglobulin approx. 725000
1474 amino acids globular protein; 4 identical subunits bound together by disulfide bonds
many endoproteases, carboxyl, thiol, serin, metallo proteases
irreversilbe inhibi tion by “trap” mecha-nism. Conforma-tional changes of α2-Macroglobulin prevents reaction of protease and substrate
5–10 mg/ml in aqueous buffer solution
A2205 Pepstatin A 685.91 acid proteases, aspartic proteases, pepsin, cathepsin D, renin, HIV- und MMTV-proteases
1–5 µM(0.7–3.5 µg/ml)
in MeOH,EtOH, acetic acid solution, DMSO
A3826 1,10-Phenanthroline monohydrate
198.24 metallo proteases, carboxy-peptidase G2 of Pseudo-monas, peptidase T, and acetylornithinase from E. coli, aminopeptidase from Apis mellifera, peptidase V from L. delbrueckii and more
complexes several metal ions (mainly zinc)
1–200 µg/ml(0.5–1 mM) or even 2 mg/ml (10 mM)
in EtOH, aceton
A2214 Phosphoramidon ( disodium salt)
543.50 (587.48)
metallo proteases, thermolysin, endothelin-converting enzyme
membrane metallo-endopeptidase inhibitor
1–10 µM in water, MeOH
A0999 PMSF*** 174.19 serin/cystein proteases, trypsin, chymotrypsin, thrombin, papain
irreversible inhibitor
0.1–1 mM 10–100 mM in EtOH1,74–17,4 mg/ml
A1799 TLCK 369.31 serin proteases, trypsin, ceras tocytin, but not chymotrypsin
specific irrever sible inhibitor
50 µg/ml 1–20 mg/ml in buffer, pH ≤ 6
A1801 TPCK 351.85 serin/cystein proteases, chymotrypsin, cerastocytin, papain, ficin, but not trypsin
irreversible inhibitor
10–100 µg/ml(28.4–284 µM)
10 mg/ml in EtOH or MeOH
A1441, A1828
Trypsin-Inhibitor ** ca. 21.5 kDa See fig. 1 trypsin, also chymotrypsin, plasmin, thrombin, plasma kallikrein
reversible serine protease inhibitor
10–100 µg/ml 1 mg/ml in water
*Inactivation by reducing agents, ** from soybean , ***The inhibition of cysteine proteases can be reverted by reducing thioles (e.g. DTT).
AbbreviationsAEBSF (4-(2-Aminoethyl)-benzolsulfonylfl uoride), APMSF (4-Amidinophenylmethanesulfonylfl uoride), E-64 (N-(trans-Epoxysuccinyl)- L-leucine-4-guanidinobutylamide), EDTA (Ethylenediaminetetraacetic acid), EGTA (Ethyleneglycol-bis-(2-aminoethyl)-tetraacetic acid), EtOH (Ethanol), MeOH (Methanol), PMSF (Phenylmethanesulfonylfl uoride), TLCK (Nα-Tosyl-L-lysine chloromethylketone), TPCK (Tosyl-L-phenylalanine chloromethylketone)
Tab. 1: Individual Protease Inhibitors (continued)
Prod.-No.
Description M [g/mol] Structure Target Protease Class,Target Enzymes
Mechanism Recommen-ded Working Concentration
Stock Solution
Prod. - No.
Description Composition Target Protease Class and Application
A7706 Protease Inhibitor Cocktail 1 Cell
AEBSF·HCl, EDTA·Na2 salt, leupeptin hemisulfate, pepstatin A
Inhibits serine, aspartic, metallo, cysteine and trypsin-like proteases. Suited in preparation of cell extracts. Lyophilized mixture to make up a 100X solution.
A7719 Protease Inhibitor Cocktail 2 MultiPurpose
AEBSF·HCl, aprotinin, E-64, EDTA·Na2 salt, leupeptin hemisulfate
Inhibits serine, metallo, cysteine and trypsin like proteases, and esterases. Lyophilized mixture of protease inhibitors against a broad spectrum of proteases from different raw materials.
A7735 Protease Inhibitor Cocktail 3 Bacteria
AEBSF·HCl, bestatin, E-64, EDTA·Na2 salt, pepstatin A
Inhibits serine, aspartic, metallo and cysteine proteases, as well as aminopeptidase B and leucine aminopeptidase. Lyophilized mixture of inhibitors of bacterial proteases.
A7757 Protease Inhibitor Cocktail 4 MammCell/Tissue Plus
AEBSF·HCl, aprotinin bestatin, E-64, leupeptin hemisulfate, pepstatin A
Inhibits serine, aspartic, cysteine and trypsin-like proteases, as well as aminopeptidase B, leucine aminopeptidase and este-rases. Concentrate of inhibitors against a broad spectrum of proteases from cells and tissue mammalian origin.
A7779 Protease Inhibitor Cocktail 5 MammCell/Tissue
AEBSF·HCl, aprotinin, E-64, leupeptin hemisulfate
Inhibits serine, cysteine and trypsin-like proteases, as well as esterases. Suited for preparation of extracts from mammlian cells and tissue.
A7802 Protease Inhibitor Cocktail 6 His-Tag Prot
AEBSF·HCl, bestatin, E-64, pepstatin A, phosphoramidon
Inhibits serine, aspartic, metallo and cysteine proteases, as well as aminopeptidase B and leucine aminopeptidase. Suited for preparation of up to 10 g of cell extracts.
Description Cat. No.
Tris ultrapure A1086
HEPES Buffer grade A1069
Urea BioChemica A1360
Guanidine hydrochloride BioChemica A1499
Magnesium chloride hexahydrate BioChemica A1036
Imidazole Buffer grade A1073
Sodium chloride BioChemica A1149
β-Mercaptoethanol BioChemica A4338
DTT BioChemica A1101
Tween® 80 BioChemica A1390
Triton® X-100 BioChemica A1388
SDS ultrapure A1112
Acrylamide 4K–Solution (30 %) A1154
Ammonium persulfate BioChemica A1142
TEMED A1148
PVDF-Star Transfer Membrane 0.45 μm A5243
Reprobe Nitrocellulose supported 0.22 μm Transfer Membrane A5237
Literature:
[1] Voet and Voet, Biochemistry, 2 nd edition. John Wiley & Sons, Inc.,New York, 1995
[2] Wang et al. (2007) MMDB: annotating protein sequences with Entrez’s 3D-structuredata base.
Nucleic Acids Res. 35 (Database issue), D298-300
[3] Chen et al. (2003) MMDB: Entrez‘s 3D-structure database. Nucleic Acids Res. 31(1),474-7
[4] Harbut et al. (2008) Development of bestatin-based activity-based probes for metallo-amino-
peptidases. Bioorg Med Chem Lett. 18, 5932-6
[5] Song and Suh (1998) Kunitz-type soybean trypsin inhibitor revisited: refined structure of its
complex with porcine trypsin reveals an insight into the interaction between a homologous inhibitor
from Erythrina caffra and tissue-type plasminogen activator.J Mol Biol. 275 (2), 347-63
Tab. 2: Protease Inhibitor Cocktails
A118,E;201101
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