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MP Protease Inhibitors

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Introduction

The presence of undesired proteases during the isolation and purification of intact peptides and proteins usually corresponds to greatly reduced yields of pure and active proteins. This is particularly true for proteins isolated from cellular extracts, since many cells and microorganisms secrete proteolytic enzymes when grown in vitro. The need for effective protease inhibition is therefore, essential for proper isolation of purified proteins.
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Mode of Action

Proteases are enzymes which direct their action upon proteins, cleaving their targets into smaller fragments consisting of polypeptides , peptides and amino acids. There are a myriad of proteases active against practically all proteins. Some proteases are quite specific , while other cleave many different proteins.
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Classification

In general , proteases are classified into two main subclasses: endopeptidases and exopeptidases. dependent upon how and where the protease cleaves the protein. these proteases act as enzymes of digestion, for repair and growth of proteins, and many other functions which ultimately require the breakdown of proteins.

Principally, proteolytic enzymes have been used to study the basic structure of proteins. Using specific proteases , a scientist can digest a native protein and analyze the fragments to determine the exact structure and sequence of the active protein.


Proteolytic Pathways Subject Protein
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Specificity

As stated earlier, proteases are generally divided into two main subclasses . The endopeptidases have the ability to cut a protein at specific positions within the molecule. They possess the ability to recognize a specific amino sequence within the complete protein and cleave the molecule at that site. A corollary may be drawn to the ability of a restriction endonuclease to recognize a specific nucleotide sequence within a DNA molecule and to cut the molecule at, or adjacent to that recognition site. Endopeptidases, however , are not usually quite as specific as a restiction enzyme in their ability to recognize only one specific site and consequently , may exhibit some non-specific, or secondary cleavage.

In contrast to endopeptidases, exopeptidases do not recognize a specific amino acid sequence within a protein . Rather, exopeptidases begin digesting the protein at one specific end of the molecule and literally chop away one amino acid at a time until they reach some specific amino acid which signals them to stop. This termination point is typically defined by both the end of the protein at which digestion begins and the precise amino acid that stops the reaction. Exopeptidases may begin digestion at either the amino or carboxyl end of the protein, depending on the actual characteristics of the specific protease. The diagram of proteolytic pathways provides a simplified illustration of the endo- and exo peptidase modes of action
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Protein Chemistry Reagents

As can be seen, proteases can be very powerful enzymes for protein digestion. While this is very useful, there is also an adverse side to this. Proteases are ubiquitous enzymes commonly found in many biological sources ranging from animal organs and tissues to microorganisms .Consequently , they often appear as contaminants in protein preparations which have been isolated from natural sources. Their presence can be a real problem in that , they may interfere with protein purification, isolation and identification techniques. In fact , the presence of a non-specific protease contaminant can wreak havoc with protein research if the protease (s) digest the protein being studied.
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Unwanted Contaminants

Many proteases are able to resist normal protein purification methods and may be difficult to inactivate. Consequently , a class of compounds called protease inhibitors has found widespread application in protein chemistry. Inhibitors are often used during protein purification to eliminate protease contamination. The choice of a suitable inhibitor can block the action of a protease without interfering with the action of the desired protein. Inhibitors may also be used by including them with a protein mixture to prevent the digestive actions of any undesired contaminating proteases.
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Inhibitor Cocktails

Since there are such large number of proteases , both specific and non-specific , as endo- and expo-peptidases, the selection of a satisfactory protease inhibitor requires careful choice. No one protease inhibitor is likely to be sufficiently effective against all proteases. As a result, it is desirable to have a ready source of protease inhibitors from which to select one that will perform under the given conditions of a specific project. Yet, often more than one contaminating protease may be present , so a single inhibitor, even when carefully selected , any not be capable of inhibiting all proteolytic activity. Consequently , the practice of preparing an inhibitor "Cocktail" of several protease inhibitors seems to be gaining favor in some areas of protein research. Again, in order to prepare an inhibitor " Cocktail" a variety of different inhibitors is highly desirable.
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MP Product Portfolio

MP is pleased to present its showcase of protease inhibitors, which include products that inhibit both specific and non-specific proteases, endopeptidases and exopeptidases . These high quality inhibitors are an integral part of any protein research project and we are placed to be able to offer such a diverse selection. Product details are listed opposite .
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General Protease Inhibitor Cocktail

A broad spectrum protease inhibitor cocktail should cover both endopeptidases and exopeptidases , non-specific and specific. The most commonly found protease classes include serine proteases, metallproteases and acid proteases. A suitable multi-purpose cocktail must contain these three classes and a broad range inhibitor for all other areas . MP has developed a broad spectrum cocktail which covers all the above requirements. The cocktail consists of four major protease inhibitors : AEBSF. Sodium EDTA. Leupeptin and Pepstain . The specificities and working concentration of these enzymes is detailed below.


InhibitorSpecificitySuggested Amount
AEBSFSerine Proteases50-250 mg/ml
EDTA-Na2Metalloproteases0.2-5 mg/ml
LeupeptinBroad Range0.5-1 mg/ml
PepstatinAcid Proteases0.5-1 mg/ml


Protease Inhibitor Cocktail Kit 158837

This convenient kit contains the following protease inhibitors in pre-weighed vials to prepare100 ml of ready-to-use inhibitor cocktail. AEBSF (40mg) . EDTA - Na2 (500 mg). Leupeptin (0.1 mg) . Pepstatin (0.1 mg.)
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References
  1. Beynon,R.J. and Bond,J.S. Proteolytic Enzymes : A Practical Approach, IRL Press . Oxford University Press. (1989).
  2. Doherty,F.J.,and Mayer,R.J., Intracellular Protein Degradiation : In Focus .IRL Press Oxford University Press. (1992)
  3. Harris,E.L.V. and Angal.S,. Protein Purification Methods : A Practical Approach. IRl Press Oxford University Press (1990)
  4. Walker,J.M.ed.. New Protein Techniques : Methods in Molecular Biology . Humana Press. (1988).
  5. Franks,F..ed.Characterization of Proteins . Humana Press (1988)
  6. Walsmann,P., Richter,M. and Markwardt,F.. Inaktivierung von Trypsin und Thrombin durch4-Amidinobenzolsulfofluorid und 4-(2-Amino-ethyl)-benzoylsulflofluorid.Acta.Biol.Med. Germ. 28. 577-579 (1972)
  7. James,G.T. Inactivation of the Protease Inhibitor Phenylmethsulfonyl Fluoride in Buffers.Anal. Biochem .. 86 574-579. (1978)
  8. Markwardt,F..Drawert.J.and Walsmann.P..Synthetic Low Molecular Weight Inhibitors of Serum Kalikrein,Biochem Pharmacol.. 23. 2247-2256. (1974)
  9. Frommer.W., et al.,J. Med.Plant..Res.. 35. 195 (1979)
  10. Umezawa,H.Methods in Enzymology. 45. 678. (1976)
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MP Product Listings

Inhibitor
Catalog Number
Specificity
Recommended Concentration
AEBSF [4-(29 Aminoethyl)- benzenesulfonyl fluoride]
193503
Serine proteases, chymotrypsin , Kallikrein (plasma) ,plasmin thrombin, trypsin
50-250 mg/ml
Amastatin
152842
199000
Binds to cell surfaces and reversibly inhibits aminopeptidases. A slow binding, competitive inhibitor of aminopeptidase M and leucine aminopeptidase
1 - 10 uM
Amidinophenyl benzoate hydrochloride
194099
A trypsin-like serine protease inhibitor
--
Amidinophenylpyruvic acid
194100
A polyvalent inhibitor of serine proteases like trypsin, factor IIa, factor Xa, and plasma kallikrein.
--
e-Amino-n- caproic acid (EACA)
101645
Inhibits carboxypeptidase B.
1-2 mM
a-1- Antichymotrypsin
191347
Specific inhibitor of chymotrypsin- like serine proteases
use at equimolar concentrations
Antipain
152843
198585
Papain, trypsin
50 mg/ml
a2-Antiplasmin
194186
It efficiently inhibits the plasminogen-activator-induced lysis of fibrin clots. Forms a covalent complex with plasmin and inactivates it.
use at equal unitage concentration to the plasmin
Antithrombin III
194105
194936
153572
Complexes with serine proteases of blood coagulation system including thrombin, plasmin, kallikrein, and factors IXa, Xa, Xla, and XIIa
Use at equimolar concentrations
a-1-Antitrypsin
191346
Most serin proteases. chymotrypsin, elastase, plasmin thrombin,trypsin Also acts on collagenase
10-15 mg/ml
p-APMSF, hydrochloride
194956
A specific inhibitor of trypsin-like serine proteases. An alternative to DFP and PMSF
Use at equimolar concentrations
Aprotinin
190382
191158
190779
Serine proteases. Inhibits chymotrypsin, Kallkrein, plasmin and trypsin. Does not inhibit thrombin.
1-2 mg/ml
Benzamidine hydrochloride
195068
Inhibitor of trypsin and trypsin-like enzymes. Inhibits factor VII autoactivation.
0.5-4.0 mM
Bestatin
152844
Amino peptidases (e.g. aminopeptidase B. luecine aminopeptidase) and other exopeptidases
40-50 mg/ml
Calpain Inhibitor I
158834
Calpains(calcium dependent cysteine protease)
15-20 mg/ml
Calpain Inhibitor II
158835
Calpains, also slightly inhibits papain
5-10 mg/ml
Calpeptin
159563
Inhibitor of calpain and cathepsin L
100 mg -1 mg/ml
Carboxypeptidase Inhibitor
194967
A potent inhibitor of a wide variety of digestive tract carboxypeptidases.1-2 mg
Chymostatin
152845
Chymotrypsins
100 mg/ml
Chymotrypsin Inhibitor I
194978
Each subunit inhibits one molecule of chymotrypsin.
10-20 ug/ml
Chymotrypsin Inhibitor II
194979
A potent inhibitor of chymotrypsin and trypsin.
0.5 - 20. ug/ug enzyme
Cystatin
194984
A competitive and reversible cysteine protease inhibitor. Inhibits papain, ficin, and cathepsin B.
Use at equimolar concentrations
Cystatin A
194985
Functions as a tight binding reversible cysteine protease inhibitor. A potent inhibitor of papain.
Use at equimolar concentrations
Cystatin C
194987
Functions as a cysteine and thiol-protease inhibitor.
Use at equimolar concentrations
DAPA
194937
Potent thrombin inhibitor
--
3,4-Dichloro- isocoumarin
158836
Serine proteases, some endopeptidases (e.g. protease, Staph.V-8). Does not inhibit papain
5-50 mg/ml
E-64
152846
Thiol proteases, papain, cathepsin B
0.5-1 mg/ml
E-64c
195985
Thiol protease inhibitor; cell impermeable
0.5-1 mg/ml
E-64d
195986
Thiol protease inhibitor; cell permeable
0.5-1 mg/ml
Ebelactone A
157894
Inhibits esterase, lipase and N-formyl methionine aminopeptidase
1-2 mg/ml
Ebelactone B
157895
See Ebalactone A
1-2 mg/ml
EDTA Sodium
195173
Inhibitor serine proteases. Also cathepsin B, papain, plasma, trypsin
0.2-0.5 mg/ml
EGTA
195174
A metalloprotease inhibitor.
1-10 mM
Elastatinal
152847
General endopeptidase inhibitor
5 mg/ml
Epiamastatin Hydrochloride
152848
A metalloprotease inhibitor with selectivity for aminopeptidases.
--
Epibestatin Hydrochloride
152849
A metalloprotease inhibitor that selectively inhibits aminopeptidases
--
Ile-Pro-Ile (Diprotin A)
153132
A reversible inhibitor of dipeptidyl peptidase IV.
10-50 mM
Lactacystin
195863
Irreversibly inhibits chymotrypsin- and tyrpsin-like proteases
--
Leupeptin
151553
Acid proteases, including cathepsin D, chymosin, pepsin, renin
0.5 mg/ml
a-2-Macroglobulin
191345
Inhibits serine proteases, chymotrysin, thrombin, trypsin. Also inhibits papain
10-100 mg/ml
Minocycline Hydrochloride
155718
Basement membrane protease inhibitor.
--
Pepstatin A
195368
Inhibitor of collagenase , thermolysin and metalloendopeptidases
0.5-1 mg/ml
1,10-Phenanthroline Monohydrate
150147
152553
A metallo-protease inhibitor.
1-10 mM
PMSF ( phenyl- methyl sulfonyl fluoride)
195381
Theonine proteases. Two distinct endopeptidase activities: Chymotrypsin- like and peptidyglutamyl- peptide hydrolase activity
15-175 mg/ml
Phosphoramidon
152851
Inhibits trypsin
5-350 mg/ml
20S Proteasome
193625
Inhibits trypsin, Also inhibits Factor Xa. plasmin and Kallikrein(plasma)
5-100 mg/ml
TLCK
152152
An irreversible inhibitor of trypsin-like serine proteases. Inactivates trypsin, but no effect on chymotrypsin.
10-100 mM
TPCK
103094
An irreversible inhibitor of chymotrypsin.
10-100 mM
Trypsin Inhibitor (chicken eggwhite)
100612
Trypsin inhibitor
10-100 mg/ml
Trypsin Inhibitor (soybean)
101113
A reversible serine protease inhibitor. Inhibits trypsin, factor Xa, chymotrypsin, kallikrein and plasmin.
10-100 mg/ml
Trypsin Inhibitor (lima bean)
100798
Inhibits trypsin. Also acts on plasmin and Kalikrein to a lesser degree
10-100 mg/ml
Val-Pro-Leu (Diprotin B)
153194
A reversible inhibitor of dipeptidyl peptidase IV.
50-100 mM
Protease
Catalog Number
SpecificityActivity
Neutralase
150208
Neutral protease most active at neutral pH. Allows isolation of double stranded DNA without use of phenol . Useful in cell culture work
1000 U/mg
Protease
101024
Crude protease from papaya, but essentially free of DNase and RNase
0.3-0.5 U/mg
Protease
150210
Excellent heat stability
50-100 U/mg
Protease
150211
Exhibits proteinase and peptidase activities. Stable in pH range 6-10 Inhibited by heavy metals, but stability is increased by Ca2-
0.7-1 U/mg
Protease
151972
Cleaves peptides at carboxyl-terminal side of either aspartate or glutamate residues. Highly specific.
ca. 500 U/mg
Protease
150209
Alkaline protease Approximately twice as active as most proteases at pH 11
15-20 U/mg
Protease
151973
Optimum pH 2.8 Essentially free of alkaline protease
0.3 U/mg
Protease
151974
Active pH 3.0
0.5 U/mg
Protease A-1
153898
Cleaves peptide bonds at carboxyl- terminal of lysine residues
4 U/mg


MP offers wide selection of additional proteases, including those listed below. Please refer to the General Catalog for complete details.


BromelainCathepsin Bg-ChymotrypsinMetalloendopeptidase
Calpain I and II Cathepsin Cd-ChymotrypsinPapain
Carboxylpeptidase ACathepsin GElastasePepsin
Carboxylpeptidase BChymotrypsinFicinProteinase K
Carboxylpeptidase Wa-Chymotrypsin KallikreinSubtilisin
Carboxylpeptidase Y b-ChymotrypsinLeucine Aminopeptidase Trypsin