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

AppliChem GmbH | Ottoweg 4 | D-64291 Darmstadt | Phone +49 6151 93 57-0 | Fax +49 6151 93 57-11 | eMail service@applichem.com | Internet www.applichem.com

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