EMD - Calbiochem - Inhibitors: Proteases

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		Technical Information

		Calbiochem Information

		Inhibitor Resource

		Protease

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Proteases: Protease Inhibitors

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Table 1: Selected Inhibitors� | Inhibitors | Related Products

The proper functioning of the cell requires an optimum level of important structural proteins, enzymes, and regulatory proteins. One mechanism whereby cells achieve this steady state level is by proteolytic degradation. Protein degradation exhibits first order kinetics and is an energy-dependent, irreversible process brought about by the action of several peptidases. Peptidases (proteases) can be subdivided into two major groups: the endopeptidases and the exopeptidases, which cleave peptide bonds either at points within the protein or remove amino acids sequentially from either the N- or C-terminus, respectively. The term proteinase is also used as a synonym for endopeptidase. The International Union of Biochemistry and Molecular Biology (IUBMB) has recognized four classes of proteases: serine proteinases, cysteine proteinases, aspartic proteinases, and metalloproteinases.

In vivo, proteins are either protected in specialized compartments or they reside in a special protective conformation. Every protein isolated from cells is a potential substrate for proteolytic enzymes. Protease inhibitors are often used in experimental protocols that require extraction and analysis of intact proteins. These inhibitors block the activity of proteases and minimize tissue damage during purification procedures and organ perfusion studies. More recently, selected protease inhibitors have been used as anti-HIV agents.

Commonly used protease inhibitors are either synthetic or of bacterial or fungal origin. They inhibit protease activity in either a reversible or irreversible manner. Over 100 naturally occurring protease inhibitors have been identified so far. They behave as tight-binding reversible or pseudo-irreversible protease inhibitors preventing substrate access to the active site through steric hindrance. Some of them act by modifying an amino acid residue of the protease active site. For example, serine proteases are inactivated by phenylmethane sulfonyl fluoride (PMSF), which reacts with the active site serine, whereas the chloromethylketone derivatives react with the histidine of the catalytic triad.

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Table 1: Selected Protease Inhibitors

Protease Inhibitor	Cat. No.	MW	Comments/Applications	Solubility	Suggested Working Concentration
Acetyl-Pepstatin	110175	643.8	An aspartyl protease inhibitor that acts as an effective inhibitor of HIV-1 proteinase (K_i = 20 nM at pH 4.7).	50% Acetic Acid	100-200 nM
AEBSF, Hydrochloride	101500	239.5	Water-soluble, non-toxic alternative to PMSF. Irreversible inhibitor of serine proteases. Reacts covalently with a component of the active site. Inhibits chymotrypsin, kallikrein, plasmin, trypsin, and related thrombolytic enzymes.	H₂O	0.1-1 mM
ALLM	208721	401.6	Inhibitor of calpain I (K_i = 120 nM), calpain II (K_i = 230 nM), cathepsin B (K_i = 100 nM), and cathepsin L (K_i = 600 pM).	Methanol, Ethanol, DMSO	0.2-2 mM
ALLN	208719	383.5	Inhibitor of calpain I (K_i = 190 nM), calpain II (K_i = 220 nM), cathepsin B (K_i = 150 nM), and cathepsin L (K_i = 500 pM). See also Cat. No. 208750.	Methanol, Ethanol, DMSO	0.2-2 mM

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Inhibitors: Protease

Product

Cat. No.

Acetyl-Pepstatin

AEBSF, Hydrochloride

ALLM

ALLN

InSolution™ ALLN

Amastatin, Streptomyces sp.

129875

e-Amino-n-caproic Acid

1381

Aminopeptidase N Inhibitor

164602

a₁-Antichymotrypsin, Human Plasma

178196

Antipain, Dihydrochloride

178223

Antipain, Hydrochloride

178220

a₂-Antiplasmin, Human Plasma

178221

Antithrombin III, Human Plasma

169756

a₁-Antitrypsin, Human Plasma

178251

p-APMSF, Hydrochloride

178281

Aprotinin, Bovine Lung, Crystalline

616370

Aprotinin, Bovine Lung, Solution

616399

Aprotinin, Bovine, Recombinant, Nicotiana sp., Animal-Free

616371

ATBI, Synthetic

189250

Benzamidine, Hydrochloride

199001

Bestatin

200484

Bestatin Methyl Ester

200485

CA-074

205530

CA-074 Me

205531

Calpastatin, Human, Recombinant, Domain I

208900

Calpeptin

03-34-0051

Carboxypeptidase Inhibitor, Potato

217359

Caspase Inhibitor, Negative Control

342000

Cathepsin B Inhibitor II

219385

Cathepsin G Inhibitor I

219372

Cathepsin Inhibitor II

219417

Cathepsin Inhibitor III

219419

Cathepsin Inhibitor I

219415

Cathepsin K Inhibitor I

219377

Cathepsin K Inhibitor II

219379

Cathepsin K Inhibitor III

219381

Cathepsin L Inhibitor I

219421

Cathepsin L Inhibitor II

219426

Cathepsin L Inhibitor IV

219433

Cathepsin L Inhibitor V

219435

Cathepsin L Inhibitor VI

219495

Cathepsin S Inhibitor

219393

Cathepsin/Subtilisin Inhibitor

219420

Chymostatin

230790

Chymotrypsin Inhibitor I, Potato

230906

Cystatin, Egg White

240891

1,5-Dansyl-Glu-Gly-Arg Chloromethyl Ketone, Dihydrochloride

251700

3,4-Dichloroisocoumarin

287815

Diisopropylfluorophosphate

30967

Dipeptidylpeptidase II Inhibitor

317621

Dipeptidylpeptidase IV Inhibitor I

416200

Dipeptidylpeptidase IV Inhibitor II

317638

E-64 Protease Inhibitor

324890

E-64, Immobilized

324891

Ecotin, E. coli

330200

EDTA, Disodium Salt, Dihydrate, Molecular Biology Grade

324503

EDTA, Tetrasodium Salt

34103

EGTA

324625

EGTA, Molecular Biology Grade

324626

Elastase Inhibitor I

324692

Elastase Inhibitor II

324744

Elastase Inhibitor III

Elastatinal

EST

FUT-175

GGACK

2-Guanidinoethylmercaptosuccinic Acid

369334

HDSF

373250

HSV Replication Inhibitor, BP5

385883

a-Iodoacetamide

407710

Kininogen, High Molecular Weight, Single Chain, Human Plasma

422686

Kininogen, High Molecular Weight, Two Chain, Human Plasma

422688

Kininogen, Low Molecular Weight, Human Plasma

422685

Leuhistin

432077

Leupeptin, Hemisulfate, Microbial

108975

a₂-Macroglobulin, Human Plasma

441251

DL-2-Mercaptomethyl-3-guanidinoethylthiopropanoic Acid

445825

a₁-PDX, Human, Recombinant, E. coli

126850

Pepstatin A, Synthetic

516481

Phenylmethylsulfonyl Fluoride

52332

Phosphoramidon, Disodium Salt

525276

PPACK II, Trifluoroacetate Salt

520219

PPACK, Dihydrochloride

520222

PPACK, Dihydrochloride, Biotinylated

520224

Prolyl Endopeptidase Inhibitor II

537011

Protease Arrest™ Reagent

539124

Protease Inhibitor Cocktail Set I

539131

Protease Inhibitor Cocktail Set II

539132

Protease Inhibitor Cocktail Set III, EDTA-Free

539134

Protease Inhibitor Cocktail Set IV

539136

Protease Inhibitor Cocktail Set V, EDTA-Free

539137

Protease Inhibitor Cocktail Set VI

539133

Protease Inhibitor Cocktail Set VII

539138

Protease Inhibitor Cocktail Set VIII

539129

Protease Inhibitor Set

539128

Serine Protease Inhibitor Cocktail Set I

565000

N^a-Tosyl-Lys Chloromethyl Ketone, Hydrochloride

616382

N^a-Tosyl-Phe Chloromethyl Ketone

616387

Tripeptidylpeptidase II Inhibitor

645905

Trypsin Inhibitor, Corn

650345

Trypsin Inhibitor, Soybean

65035

Trypsin Inhibitor, Soybean, High Activity

650357

D-Val-Phe-Lys Chloromethyl Ketone, Dihydrochloride

627624

(Z-LL)₂ Ketone

421050

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Other Related Products

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Protease Inhibitor Cocktail Set I, Cat. No. 539131

A cocktail containing five protease inhibitors that will inhibit a broad range of proteases. Reconstitute each vial with 1 ml H₂O to obtain a 100x stock solution. When diluted, 1x stock solution contains the following amount of inhibitors.

Product	Cat. No.	Mol. Wt.	1x Concentration	Target Protease
AEBSF, Hydrochloride	101500	239.5	500 mM	Serine Proteases
Aprotinin	616370	6512	150 nM	Serine Proteases and Esterases
E-64 Protease Inhibitor	324890	357.4	1 mM	Cysteine Proteases
EDTA, Disodium	324503	372.2	500 mM	Metalloproteases
Leupeptin, Hemisulfate	108975	475.6	1 mM	Cysteine Proteases and Trypsin-like proteases

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Protease Inhibitor Cocktail Set II, Cat. No. 539132

This cocktail is recommended for use with bacterial cell extracts. Cocktail contains five protease inhibitors with broad specificity for the inhibition of aspartic, cysteine, serine, and metalloproteases as well as aminopeptidases. Reconstitute each vial with 1 ml DMSO and 4 ml H₂O to obtain a 5 ml stock solution. When reconstitued each vial will contain the following amount of inhibitors. Note: 1 set = 1 vial of lyophilized protease inhibitor cocktail and 1 vial DMSO, 1 ml.

Product	Cat. No.	Mol. Wt.	Concentration in the Vial	Target Protease
AEBSF, Hydrochloride	101500	239.5	20 mM	Serine Proteases
Bestatin	200484	308.4	1.7 mM	Aminopeptidase B and Leucine Aminopeptidase
E-64 Protease Inhibitor	324890	357.4	200 mM	Cysteine Proteases
EDTA, Disodium	324503	372.2	500 mM	Metalloproteases
Pepstatin A	516481	685.9	2 mM	Aspartic Proteases

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Protease Inhibitor Cocktail Set III, Cat. No. 539134

This cocktail is recommended for use with mammalian cells and tissue extracts. Cocktail contains six protease inhibitors (in 1 ml of DMSO) with broad specificity for the inhibition of aspartic, cysteine, and serine proteases as well as aminopeptidases. Each vial contains the following amount of inhibitors. One ml is sufficient for 20 g of tissue.

Product	Cat. No.	Mol. Wt.	Concentration in the Vial	Target Protease
AEBSF, Hydrochloride	101500	239.5	100 mM	Serine Proteases
Aprotinin, Bovine Lung, Lyophilized	616398	6512	80 mM	Broad Spectrum, Serine Proteases
Bestatin	200484	308.4	5 mM	Aminopeptidase B and Leucine Aminopeptidase
E-64 Protease Inhibitor	324890	357.4	1.5 mM	Cysteine Proteases
Leupeptin, Hemisulfate	108975	475.6	2 mM	Cysteine Proteases and Trypsin-like Proteases
Pepstatin A	516481	685.9	1 mM	Aspartic Proteases

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Protease Inhibitor Cocktail Set IV, Cat. No. 539136

This cocktail is recommended for fungal and yeast cell extracts. Cocktail contains four protease inhibitors (in 1 ml of DMSO) with broad specificity for the inhibition of aspartic-, cysteine-, metallo-, and serine-proteases. Each vial contains the following amount of inhibitors.

Product	Cat. No.	Mol. Wt.	Concentration in the Vial	Target Protease
AEBSF, Hydrochloride	101500	239.5	100 mM	Serine Proteases
E-64 Protease Inhibitor	324890	357.4	1.5 mM	Cysteine Proteases
Pepstatin A	516481	685.9	2 mM	Aspartic Proteases
o-Phenanthroline	516705	198.2	500 mM	Metalloproteases

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Protease Inhibitor Cocktail Set V, EDTA-Free, Cat. No. 539137

A cocktail containing four protease inhibitors for the inhibition of serine, cysteine, but not metalloproteases. Reconstitute each vial with 1 ml H₂O to obtain a 100x stock solution. When diluted to 1x stock solution, the set will contain the following amount of inhibitors.

Product	Cat. No.	Mol. Wt.	1x Concentration	Target Protease
AEBSF, Hydrochloride	101500	239.5	500 mM	Serine Proteases
Aprotinin, Bovine Lung, Crystalline	616370	6512�	150 nM	Broad Spectrum, Serine Proteases
E-64 Protease Inhibitor	324890	357.4	1 mM	Cysteine Proteases
Leupeptin, Hemisulfate	108975	475.6	1 mM	Cysteine Proteases and Trypsin-like Proteases

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Protease Inhibitor Cocktail Set VI, Cat. No. 539133

This cocktail is recommended for use with plant cell extracts. Cocktail contains six protease inhibitors (in 1 ml DMSO) with broad specificity for the inhibition of aspartic, cysteine, serine, and metalloproteases as well as aminopeptidases. Each vial contains the following amount of inhibitors. One ml is recommended for 30 g various plant tissues.

Product	Cat. No.	Mol. Wt.	Concentration in the Vial	Target Protease
AEBSF, Hydrochloride	101500	239.5	200 mM	Serine Proteases
Bestatin	200484	308.4	10 mM	Aminopeptidase B and Leucine Aminopeptidase
E-64 Protease Inhibitor	324890	357.4	3 mM	Cysteine Proteases
Leupeptin, Hemisulfate	108975	475.6	2 mM	Cysteine Proteases and Trypsin-like Proteases
o-Phenanthroline	516705	198.2	500 mM	Metalloproteases
Pepstatin A	516481	685.9	2 mM	Aspartic Proteases

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Protease Inhibitor Cocktail Set VII, Cat. No. 539138

This cocktail is recommended for purification of proteins containing His•Tag� sequences. Cocktail contains five protease inhibitors (in 1 ml DMSO) with broad specificity for the inhibition of cysteine, serine, aspartic, and thermolysin-like proteases and aminopeptidases. Each vial contains the following amount of inhibitors. One ml is recommended for the inhibition of proteases in 10 g cells.

Product	Cat. No.	Mol. Wt.	Concentration in the Vial	Target Protease
AEBSF, Hydrochloride	101500	239.5	100 mM	Serine Proteases
Bestatin	200484	308.4	5 mM	Aminopeptidase B and Leucine Aminopeptidase
E-64 Protease Inhibitor	324890	357.4	1.5 mM	Cysteine Proteases
Pepstatin A		685.9	2 mM	Aspartic Proteases
Phosphoramidon	525276	587.5	200 mM	Metalloendopeptidases

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Protease Inhibitor Cocktail Set VIII, Cat. No. 539129

A DMSO solution of three protease inhibitors with selective specificity for the inhibition of cysteine proteases, including calpains, cathepsins, and papain. Each vial contains the following amount of inhibitors.
Product
Cat. No.

Mol. Wt.

Concentration in the Vial

Target Protease

ALLN
208719
383.5�
1.56 mM
Calpain I/II, Cathepsin B,
Cathepsin L, Cysteine Proteases

Cathepsin Inhibitor I
219415
475.5
500 mM
Cathepsin B, Cathepsin L,
Cathepsin S, Papain

E-64 Protease Inhibitor
324890
357.4
1.5 mM
Cysteine Proteases

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Protease Arrest™ Reagent, Cat. No. 539124

An optimized concentration of various reversible and irreversible inhibitors to inhibit serine, cysteine, and calpain proteases. Suitable for the protection of proteins purified from animal tissues, plant tissues, yeast, and bacteria. Protease Arrest™ Reagent is provided as a 50x solution that when diluted in extraction buffer at pH 7.0 - 8.0 inhibits 95 - 98% of protease activity. EDTA is also provided separately to inhibit metalloproteinases.

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Serine Protease Inhibitor Cocktail Set I, Cat. No. 565000

A cocktail of four protease inhibitors that is useful for inhibition of a broad range of serine proteases. Reconstitute each vial with 1 ml of H₂O to obtain a 100x stock solution. Each vial contains the following amount of inhibitors.
Product
Cat. No.

Mol. Wt.

Concentration in the Vial

Target Protease

AEBSF, Hydrochloride
101500
239.5
500 mM
Serine Proteases

Aprotinin, Bovine Lung, Lyophilized
616398
6512
420 nM

Elastatinal
324691
512.6�
20 mM
Serine Proteases

GGACK, Dihydrochloride
347436
392.8
1 mM

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