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AppliCations No.14 Protease Inhibitors Proteases 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 activity, 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 synthesized in the laboratory or purified from natural sources. Keywords Protein isolation Protease activity competitive inhibition specific protease inhibitor Fig.1 Complex between trypsin inhibitor from soybean (a Kunitz-type proteinase 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). 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 proteinprotein interaction to the active site of trypsin (Fig. 1) and related serine proteases, (iii) α2-Macroglobulin traps endopeptidases 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 i mportance 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). Tab. 1: Individual Protease Inhibitors Prod.No. Description M [g/mol] Structure Target Protease Class, Target Enzymes Mechanism Recommended 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 10–50 µM 1–5 mM in water A2132 Aprotinin 6511.52 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, aminopeptidase B, leucine aminopeptidase, tripeptide aminopeptidase, 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 basic protein, consists of 58 amino acids irreversible inhibitor of plasma serine proteases (trypsin-like proteases) serine proteases, trypsin, chymotrypsin, kallikrein, plasmin Tab. 1: Individual Protease Inhibitors (continued) Prod.No. Description M [g/mol] Structure A2097 Iodoacetic acid* sodium salt 207.93 serine proteases A2183 Leupeptin hemisulfate 475.60 serine/cysteine proteases, plasmin, trypsin, papain, cathepsin B, thrombin, calpain reversible inhibitor A2186 α 2-Macroglobulin approx. 725000 many endoproteases, carboxyl, thiol, serin, metallo proteases irreversilbe inhibition by “trap” mechanism. Conformational changes of α2-Macroglobulin prevents reaction of protease and substrate A2205 Pepstatin A 685.91 acid proteases, aspartic proteases, pepsin, cathepsin D, renin, HIV- und MMTV-proteases A3826 1,10-Phenanthroline monohydrate 198.24 metallo proteases, carboxypeptidase G2 of Pseudomonas, peptidase T, and acetylornithinase from E. coli, aminopeptidase from Apis mellifera, peptidase V from L. delbrueckii and more A2214 Phosphoramidon (disodium salt) 543.50 (587.48) A0999 PMSF*** A1799 1474 amino acids globular protein; 4 identical subunits bound together by disulfide bonds Target Protease Class, Target Enzymes Mechanism Recommended Working Concentration Stock Solution 1–5 mM (208–1040 µg/ml) in aqueous solution 5–50 µg/ml (10–100 µM) 1 mg/ml in aqueous solutions 5–10 mg/ml in aqueous buffer solution 1–5 µM (0.7–3.5 µg/ml) in MeOH,EtOH, acetic acid solution, DMSO complexes several metal ions (mainly zinc) 1–200 µg/ml (0.5–1 mM) or even 2 mg/ml (10 mM) in EtOH, aceton metallo proteases, thermolysin, endothelin-converting enzyme membrane metalloendopeptidase inhibitor 1–10 µM in water, MeOH 174.19 serin/cystein proteases, trypsin, chymotrypsin, thrombin, papain irreversible inhibitor 0.1–1 mM 10–100 mM in EtOH 1,74–17,4 mg/ml 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 ** trypsin, also chymotrypsin, plasmin, thrombin, plasma kallikrein reversible serine protease inhibitor 10–100 µg/ml 1 mg/ml in water ca. 21.5 kDa See fig. 1 *Inactivation by reducing agents, ** from soybean , ***The inhibition of cysteine proteases can be reverted by reducing thioles (e.g. DTT). Abbreviations AEBSF (4-(2-Aminoethyl)-benzolsulfonylfluoride), APMSF (4-Amidinophenylmethanesulfonylfluoride), E-64 (N-(trans-Epoxysuccinyl)L-leucine-4-guanidinobutylamide), EDTA (Ethylenediaminetetraacetic acid), EGTA (Ethyleneglycol-bis-(2-aminoethyl)-tetraacetic acid), EtOH (Ethanol), MeOH (Methanol), PMSF (Phenylmethanesulfonylfluoride), TLCK (Nα-Tosyl-L-lysine chloromethylketone), TPCK (Tosyl-L-phenylalanine chloromethylketone) Tab. 2: Protease Inhibitor Cocktails Prod.- Description No. Composition Target Protease Class and Application A7706 Protease Inhibitor Cocktail 1 Cell AEBSF·HCl, EDTA·Na2 salt, l eupeptin 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 esterases. 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. Related Products: Description Cat. No. Tris ultrapure A1086 HEPES Buffer grade A1069 Urea BioChemica A1360 Guanidine hydrochloride BioChemica A1499 Magnesium chloride hexahydrate BioChemicaA1036 Imidazole Buffer grade A1073 Sodium chloride BioChemica A1149 β-Mercaptoethanol BioChemica A4338 DTT BioChemica A1101 Tween® 80 BioChemica A1390 A1388 Triton® X-100 BioChemica 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-aminopeptidases. Bioorg Med Chem Lett. 18, 5932-6 [5]Song and Suh (1998) Kunitz-type soybean trypsin inhibitor revisited: refined structure of its c omplex 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 AppliChem GmbH | Ottoweg 4 | D-64291 Darmstadt | Phone +49 6151 93 57-0 | Fax +49 6151 93 57-11 | eMail [email protected] | Internet www.applichem.com A118,E;201101 4t Matthes + Traut · Darmstadt