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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
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