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Resources: Protease Inhibitors

The following table gives an overview on the use and application of frequently used protease inhibitors in biochemistry and cell biology. It summarizes information on the mechanism of action, target protease class, solubility, concentration, and lists corresponding references.

Protease Inhibitors M.W. Description/Specificity of Inhibitor Solubility
Stability		 Concentration
Range a)		 References
AEBSF-HCl 239.7 Irreversible inhibitor of Thrombin and other serine proteases. Inhibits by acylation of the active site of the enzyme. Much less toxic than PMSF and DFP H2O, Aqueous solutions are stable between pH 5-6 0.1 - 2mM 60,83,84
Amastatin-HCl 511 Non-toxic reversible metallo-protease inhibitor. Inhibits many membrane-bound peptidases which are critical regulators of peptide hormones, e.g. aminopeptidase A and M, but not aminopeptidase B. Inhibits also leucine aminopeptidase. Ethanol
 0.5 % AcOH		 1 - 100 µM 1,2,3,4
(epsilon)-Aminocaproic acid 131.2 Highly active inhibitor of fibrinolysin and chymotrypsin. H2O 1 - 20 mM 5,6
(alpha)1-Antichymotypsin from human plasma 68000 Glycoprotein that inhibits chymotrypsin-like proteases (above all human neutrophil cathepsin G) by forming stable complexes. Acute phase protein; concentration in plasma increases after events like inflammation or tissue damage Aqueous buffers Used at equimolar concentration 7,8,9,10
Antipain-HCL 678.2 Reversible inhibitor of serine and cysteine proteases. Inhibits papain and trypsin more specificity than leupeptin. Plasmin is inhibited only slightly. Also involved in inhibition of RNA synthesis H2O
Methanol DMSO		 1 - 100 µM 11,12,13
Antithrombin III from human plasma ca. 60000 Glycoprotein that plays a major role in controlling serine proteases in the blood clotting cascade. Inactivates above all thrombin by forming an extremely stable complex, an effect which is enhanced by heparin. Inhibits also other proteases of the coagulation cascade like plasmin, kallikrein, factor IXa, Xa, XIa and XIIa. H2O Used at equimolar concentrations 8,14 15,16
(alpha)1-Antitrypsin from human plasma ((alpha)1-proteinase inhibitor) ca. 53000 Glycoprotein that is mainly involved in the control of neutrophil elastase activity. Inhibits also most of other mammalian serine proteases but at a lower rate. Blocks the action of target enzymes by binding nearly irreversibly to their active site. H2O
Aqueous buffers

 

 Used at equimolar concentrations 8,9,17,18,19
APMSF-HCl
(4-Amidinophenyl-methane
sulfonyl-fluoride)		 252.7 Irreversible inhibitor of trypsin-like serine proteases. Stronger inhibitor than PMSF, but does not inhibit chymotrypsin and acetylcholine esterase. H2O
(20 mg/ml).
Aqueous solutions are stable when stored in aliquots at -20 °C		 10-50 µM 20,21
Aprotinin (Trypsin inhibitor from bovine lung) ca. 6500 Basic single-chain polypeptide that inhibits numerous serine proteases by binding to the active site of the enzyme, forming tight complexes. It inhibits above all plasmin, kallikrein, trypsin, chymotrypsin and urokinase, but not carboxypeptidase A and B, papain, pepsin, subtilisin, thrombin and factor X. Used in cell culture to prevent proteolytic damage to cells  and to extend lifetime of cells. H2O, Aqueous buffers. Sterile filtered solutions at pH 5-8 are stable for several months. Denatures at pH > 12 In cell culture: 0.01 - 3 µg/ml; in other applications: 10 - 250 µg/ml 22,23,24,25
Arphamenine A 387.4 Inhibitor of the metallo-protease Aminopeptidase B H2O, MeOH IC50:
0.006 µg/ml		 85,86
Arphamenine B 403.4 Inhibitor of the metallo-protease Aminopeptidase B H2O, MeOH IC50:
0.002 µg/ml		 85,86
Benzamidine-HCl 174.6 Potent inhibitor of thrombin and trypsin H2O 0.1 - 50 mM 26,27,28
Bestatin-HCl 344.8 Metalloprotease inhibitor with multi-pharmacological functions. Inhibits cell surface aminopeptidases (notably B) and leucine aminopeptidase. Inhibitor of leukotriene A4 hydrolase and of enkephalin degradation in cell preparations from brain. Has anticarcinogenic and immunomodulating properties. Methanol
(5 mg/ml)		 1 - 150 µM
Mitogenic effects at nmolar concentrations		 29,30,31,32,33
CA-074 383.4 Inhibitor of Cathepsin B DMSO 0.01 - 1 µM 87,88,89
CA-074-Me 397.5 Proinhibitor for intracellular Cathepsin B. Membrane-permeable analog of CA-074 DMSO 1 µM 90
Calpain Inhibitor I 367.2 Tripeptide aldehyde. Specific inhibitor of the Ca2+-dependent cysteine protease calpain I and of cathepsin B and L. Ethanol,
Methanol, DMF, DMSO (10 mg/ml)
 1 - 50 µM 34,35,36
Calpain Inhibitor II 385.2 Tripeptide aldehyde. Specific inhibitor of the Ca2+-dependent cysteine protease calpain II and of cathepsin B and L. Ethanol, Methanol, DMF, DMSO
(10 mg/ml)		 1 -50 µM 35,36,37,38
Cathepsin Inhibitor Z-Phe-Gly-NHO- Bz-pMe 489.5 Specific inhibitor of Cathepsin B/L/S and Papain DMSO, EtOH, Acetonitrile Ki:
0.15 - 16 µM		 91,92
Chymostatin ca. 600 Peptide-derived aldehyde (mixture of 3 components). Reversible inhibitor of chymotrypsin-like serine and some cysteine proteases DMSO
Acetic acid		 10 - 100 µM 11,39,40,41
DFP
(Diisopropylfluoro-phosphate)		 184.2 A potent irreversible inhibitor of serine proteases and acetyl choline esterase. Highly toxic! Isopropanol; aqueous solutions are unstable 10 - 100 µM 28,42,43
Dipeptidylpeptidase IV Inhibitor
H-Glu-(NHO-Bz)Pyr		 354.8 Reversible inhibitor of Dipeptidylpeptidase IV/CD26 and Prolylendopeptidase DMSO, EtOH, H2O Ki:
1 µM		 93
Diprotin A 341.5 Reversible inhibitor of the metallo-protease Dipeptidylaminopeptidase IV H2O Ki:2.2 µM
 94
E-64 357.4 Non-competitive irreversible inhibitor of papain and other cysteine proteases. Forms a thioether bond with the sulfhydryl group in the active center of the enzyme. Useful for active site titration: one mole of E-64 inhibits one mole of protease. H2O, DMSO Mixtures of water and ethanol 1 - 10 µM 51,52,53
E-64d (EST) 342.4 Membrane-permeable analog of E-64c DMSO 1 µM 90,99,100
Ebelactone A 338.5 Non-toxic inhibitor for esterases, acylpeptide hydrolase, lipase and N-formylmethionine aminopeptidase Methanol (200 mg/ml) 1 - 10 µM 43,44,45
Ebelactone B 352.5 Non-toxic inhibitor for esterase, lipase and N-formyl-methionine aminopeptidase. Inhibits also carboxypeptidase Y-like   exopeptidase. Methanol (200 mg/ml) 1 - 10 µM 44,46,47
EDTA-Na2 372.3 Reversible inhibitor of metalloproteases H2O (pH 8) 1 - 10 mM 48,49
EGTA 380.4 Inhibits metalloproteases.. Reveals high selectivity for Ca2+ over Mg2+ ions. NH4OH, NaOH 1 - 10 mM 50
Elastatinal 512.6 Inhibitor of Elastase H2O, MeOH, DMSO Ki: 0.21 µM
(with Ac-Ala-Ala-Ala-OMe as substrate)		 11
Hirudin 7027 Inhibits thrombin by blocking substrate binding groups H2O
Aqueous buffers		 Used at equimolar concentrations 54,55
Leuhistin 241.3 Inhibitor of Aminopeptidase M H2O, DMSO,
EtOH, MeOH		 IC50:
0.2 µg/ml		 102,103,104
Leupeptin-
hemisulfate		 475.6 Tripeptide aldehyde. Reversible competitive inhibitor of serine and cysteine proteases. Inhibits also phospholipase D and C activation in rat hepatocytes. H2O
Stable for sereval months when stored in aliquots at -20 °C		 1 - 100 µM 11,56,57,58
(alpha)2-Macroglobulin from human plasma 725000 Glycoprotein composed of 4 identical subunits. Broad-range endoproteinase inhibitor. Inhibits by forming a »trap« around the enzyme allowing only small substrate molecules to enter and to be cleaved by the entrapped protease. H2O
Stable for several months when stored in aliquots at -20 °C		 Used at equimolar concentrations 8,59
PEFABLOC® SC b)
4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride		 239.5 Water-soluble and relatively non-toxic irreversible inhibitor of thrombin and other serine proteases. Inhibits by acylation of the active site of the enzyme. H2O
(20 g/100 ml) Stable for several months between pH 5 - 6; limited stability above pH 7.5		 0.1 - 5 mM in cell culture: 0.1 - 0.25 mM 60,61,62
Pepstatin A 685.9 Pentapeptide derivative. Reversible inhibitor of aspartic proteases, e.g. pepsin, cathepsin D, chymosin, renin Methanol
(1 mg/ml) DMSO		 1 - 10 µM 11,63,64,65
Phebestin 441.5 Inhibitor of Aminopeptidase N H2O, DMSO, MeOH IC50:
0.18 µg/ml		 105
PMSF
Phenylmethyl sulfonyl fluoride		 174.2 Irreversibly inhibits serine proteases by sulfonylation of the serine residue in the active site of the protease. Inhibits also papain (reversible by DTT treatment) and acetylcholin-esterase. Does not inhibit metallo-, aspartic- and most cysteine proteases. Isopropanol, ethanol, methanol. (100 - 200mM) Unstable in aqueous solution 0.1 - 1 mM 66,67,68
Phosphoramidon 587.5 Specific inhibitor of thermolysin and neutral endopeptidase-24.11 (ANP Degradation Enzyme). Inhibits also the activity of Endothelin Converting Enzyme, collagenase and metallo-endoproteinases from many microorganisms. Does not inhibit serine, cysteine and aspartic proteases H2O
(20 mg/ml) DMSO Methanol		 1 - 100 µM 11,69,70,71,72
TLCK
(1-Chloro-3-tosylamido-7-amino-2-heptanone HCl)		 369.3 Irreversibly inhibits trypsin but not chymotrypsin by alkylating  the histidine residue in the active site of the enzyme. Inhibits also some other serine and cysteine proteases like bromelain, ficin and papain. TLCK does not react with zymogens or in active protease-inhibitor complexes. 1 mM HCl, DMSO H2O
(20 mg/ml). Aqueous solutions are unstable above pH 7		 10 - 1000 µM 73,74,75,76
TPCK
(1-Chloro-3-tosylamido-4-phenyl-2-butanone)		 351.8 Irreversibly inhibits chymotrypsin but not trypsin by specifically reacting with histidine. Inhibits also other serine and cysteine protease such as bromelain, ficin and papain. Ethanol (20 mg/ml) sparingly soluble in water; unstable at alkaline pH 10 - 1000 µM 62,77
Trypsin inhibitor from egg white
(Ovomucoid)		 ca. 28000 Monomeric Glycoprotein. Inhibits bovine (but not human) trypsin in a 1:1 molar ratio. Inhibition is reversible and pH dependant. H2O, 1 mM HCl
(1 mg/ml) Very stable between pH 3 - 7 against heat and 9 M urea. Unstable at alkaline pH		 Used at equimolar concentrations
(10-100 µg/ml)		 78,79,80
Trypsin inhibitor from soybean ca. 22000 Monomeric protein. Reversible serine protease inhibitor. Inhibits trypsin, factor Xa, plasmin and plasma kallikrein, but not tissue kallikrein. H2O
(1 mg/ml) Sensitive to heat and high pH		 Used at equimolar concentrations (10-100 µg/ml) 81,82


a) Concentration range refers to data frequently used in the literature. The optimal concentration depends very much on the test system under investigation and has to be determined in each case empirically.
b) PEFABLOC is a registered trademark of Pentapharm/Basel

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