Biomedical Engineering Reference
In-Depth Information
8.5 Protease Inhibitors
In the literature there are a lot of more or less specific inhibitors of
proteases. During daily work with tissue homogenates, a mixture
of inhibitors with broad specificity has been proved with success.
This mixture is given in Table 8.12.
The individual inhibitors are made as stock solutions, mostly
1000-fold concentrated with respect to final dilution. Add the stock
solutions immediately before starting homogenization.
0.1 M PMSF
dissolve 87 mg into 5 ml i-propanol or ethanol;
Inhibitor Stock
Solutions
stable for 4 - 6 weeks when refrigerated
1 M Iodoacetamide dissolve 925 mg in 5 ml ddH
2
O; stable at RT
0.5 M EDTA
dissolve 1.86 g EDTA, disodium salt, in about
8 ml ddH
2
O,adjustpH8withsodiumhydrox-
ide and fill up to 10.0 ml;stableatRT
Leupeptin
dissolve
2.5 mg in
5 ml
ddH
2
O;
stable
for
1 week when refrigerated
0.1 M Benzamidine dissolve 87 mg in 5 ml ddH
2
O; stable at RT
0.1 M o-Phenanthroline dissolve 99 mg in about 4 ml ddH
2
O, ad-
just pH 6 with hydrochloric acid, fill up to
5.0 ml;stableatRT
/
ml in methanol; stable at −20
◦
C for sev-
eral weeks
Pepstatin A
1 mg
µ
/
Further inhibitors (working concentrations in
g
ml in parenthe-
sis) are:
4-(2-aminoethyl)-benzensulfonyl fluoride (100 - 1000; Pefabloc
SC, AEBSF, use instead of PMSF or 4-amidinophenyl-methylsulfonyl
fluoride (10 - 40; APMSF); antipain (50); aprotinin (0.06 - 2); besta-
ine (40); calpain inhibitor I (17; N-acetyl-Leu-Leu-neulorcinal); N-
[N-l-3-trans-carboxiran-2-carbonyl)-l-leucyl]-4-aminobutyl gua-
nidin (0.5 - 10; E-64); l-1-chloro-3-(4-tosylamido)-7-amino-2-
heptanone (37 to 50; TLCK); l-1-chloro-3-(4-tosylamido)-4-phen-
yl-2-butanone (70 to 100; TPCK); chymostatin (6 - 60; N-(
α
-rham-
Table 8.12.
Protease inhibitor mix
Inhibitor
Final
Specificity
concentration
Benzamidine
0.1 mM
Trypsin and similar proteases
EDTA
5 mM
Metalloproteases
Iodacetamide
1 mM
Thiol proteases
µ
/
Leupeptin
0.5
g
ml
(broad specificity)
µ
/
Pepstatin A
0.5
g
ml
Acidic proteases
o-Phenanthroline
0.1 mM
(broad specificity)
PPSF
0.1 mM
Serine and thiol proteases
