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Protease Inhibitor & Reduction Agents
AEBSF / 6-Aminocaproic acid / APMSF / Aprotinin / Benzamidine / Bestatin / Chymostatin
/ E-64 / EDTA / EGTA / Leupeptin / Pepstatin A / PMSF
TCEP / DTE / DTT / 2-Mercaptoethanol
.....
protease (also called a peptidase or proteinase) is an enzyme that performs proteolysisprotein catabolism by hydrolysis of peptide bonds.
Proteases have 
evolved multiple times, and different classes of protease can perform the same reaction by completely different catalytic mechanisms. Proteases can be found in AnimaliaPlantaeFungiBacteriaArchaea and viruses.

By splitting the peptide bonds that link amino acid residues, the proteases are involved in digesting long protein chains into shorter fragments. Some detach the terminal amino acids from the protein chain (exopeptidases, such as aminopeptidasescarboxypeptidase A); others attack internal peptide bonds of a protein (endopeptidases, such as trypsinchymotrypsinpepsinpapainelastase).   
Proteases are also secreted to process food, e.g. in the intestinal tract of animals.
https://en.wikipedia.org/wiki/Protease#Enzymatic_function_and_mechanism.   


Protease inhibitor

In biology and biochemistry, protease inhibitors are molecules that inhibit the function of proteases (enzymes that aid the breakdown of proteins). Many naturally occurring protease inhibitors are proteins.

In medicine, protease inhibitor is often used interchangeably with alpha 1-antitrypsin (A1AT, which is abbreviated PI for this reason).[1] A1AT is indeed the protease inhibitor most often involved in disease, namely in alpha-1 antitrypsin deficiency.
https://en.wikipedia.org/wiki/Protease_inhibitor_(biology)

Protease Inhbitor s are abundant in every organism inside their cells and here fulfill a crucial role in regulating the life cycle of proteins, activating pro-enzymes or eliminating problematic proteins.

Protease Inhibitors in protein purification procedures
 
Whenever proteins are analyzed in biological samples or purified from a natural source, protease activity is a potential threat.
During sample preparation, cells are frequently lysed and in this way they set free high amounts of protease activities that may digest the proteins of interest.
The days of work for cell culture and protein sample preparation can be destroyed within a few seconds.
Labs generally apply two basic strategies to knock out such unwanted proteolytic activities:
(a) Cooling the sample or cell lysate, and
(b) Adding chemical inhibitors of proteases.
The most common proteases are the serine proteases Chymotrypsin, Kallikrein, Plasmin, Proteinase K, Thrombin and Trypsin. Hence their deactivation after cellular homogenization is very important in the isolation of proteins in order to ensure satisfactory protein purification yields.    
Our team partner, the Biosynth offers most of the successfully used protease inhibitors that offer both high purity and the requisite grade for use in biochemistry laboratories and production scale purification process: 
 
4-(2-Aminoethyl)-benzenesulfonylfluoride hydrochloride (AEBSF)
 https://en.wikipedia.org/wiki/AEBSF
Target Enzymes:
Serine Proteases
AEBSF or 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride is a water-soluble, irreversible serine protease inhibitor with a molecular weight of 239.5 Da. It inhibits proteases like chymotrypsinkallikreinplasminthrombin, and trypsin. The specificity is similar to the inhibitor PMSF, nevertheless AEBSF is more stable at low pH values. Typical usage is 0.1 - 1.0 mM.

Mechanism of action:
Both AEBSF and 
PMSF are sulfonyl fluorides and are sulfonylating agents.[1]Sulfonyl fluorides act by reacting with the hydroxy group of the active site serine residue to form a sulfonyl enzyme derivative. This derivative may be stable for long periods of time except at high pH.[2]AEBSF  are irreversible serine protease inhibitors and are, thus, part of most homogenization buffers and added to cell lysates.
 Deactivation (irreversible inhibition) occurs through esterification of the serine hydroxyl group.
While PMSF not only deactivates serine proteases but also any other enzyme that contains serine in its active site, and therefore cannot be used if the biological activity of such an enzyme needs to be maintained, the AEBSF is often preferred over PMSF or DFP  (diisopropylfluorophosphate), because it is less toxic and water soluble.
Aqueous AEBSF solutions are stable at slightly acidic pH values.
 
6-Aminocaproic acid
 https://en.wikipedia.org/wiki/Aminocaproic_acid
 Target Enzymes: Serine Proteases
Aminocaproic acid (also known as ε-aminocaproic acid, ε-Ahx, or 6-aminohexanoic acid) is a derivative and analogue of the amino acid lysine, which makes it an effective inhibitor for enzymesthat bind that particular residue. Such enzymes include proteolytic enzymes like plasmin, the enzyme responsible for fibrinolysis. For this reason it is effective in treatment of certain bleeding disorders, and it is marketed as Amicar. Aminocaproic acid is also an intermediate in the polymerization of Nylon-6, where it is formed by ring-opening hydrolysis of caprolactam. 
 
4-Amidinophenylmethansulfonylfluorid - Hydrochlorid (APMSF)
 https://www.biosynth.com/products/products/A-4895.html
https://en.wikipedia.org/wiki/PMSF

 Target Enzymes:
Serine Proteases, Trypsin, Thrombin, Factor Xa, Plasmin.
Irreversiblely inhibit serine proteases specifically for lysine or arginine substrate.
Also new discovered proteases characterizations. 
 
Aprotinin
 https://en.wikipedia.org/wiki/Aprotinin#Chemistry
Target Enzymes:
Plasmin, Kallikrein, Trypsin, Chymotrypsin
The drug aprotinin (Trasylol, previously Bayer and now Nordic Group pharmaceuticals), is a small protein bovine pancreatic trypsin inhibitor (BPTI), or basic trypsin inhibitor of bovine pancreas, which is an antifibrinolytic molecule that inhibits trypsin and related proteolytic enzymes.
In vitro use : Small amounts of aprotinin can be added to tubes of drawn blood to enable laboratory measurement of certain rapidly degraded proteins such as 
glucagon.
In cell biology aprotinin is used as an enzyme inhibitor to prevent protein degradation during lysis or homogenization of cells and tissues.
Aprotinin can be labelled with fluorescein isothiocyanate. The conjugate retains its antiproteolytic and carbohydrate-binding properties
[21]and has been used as a fluorescent histochemical reagent for staining glycoconjugates (mucosubstances) that are rich in uronic or sialic acids.[22]
BPTI is one of the most thoroughly studied proteins in terms of structural biology, experimental and computational dynamics, mutagenesis, and folding pathway. It was one of the earliest protein crystal structures solved, in 1970 in the laboratory of Robert Huber,[28] and was the first protein to have its structure determined by NMR spectroscopy, in the laboratory of Kurt Wuthrich at the ETH in Zurich in the early 1980s.[29][30]                                 
Aprotinin from bovine lung :  Aprotinin is a small protein but a powerful inhibitor that prevents activity of several serine proteases (trypsin, chymotrypsin, plasmin, and kallikrein) already at low inhibitor concentrations.      
 
Benzamidine hydrochloride monohydrate (Benzamidine)
https://en.wikipedia.org/wiki/Benzamidine
Target Enzymes:
Serine Proteases
Benzamidine is a reversible competitive inhibitor of trypsin, trypsin-like enzymes and serine proteases.It is often used as a ligand in protein crystallography to prevent proteases from degrading a protein of interest; the triangular diamine group at the bottom gives it a very obvious 'stick-man' shape which shows up in difference density maps. The benzamidine moiety is also found in some pharmaceuticals, like dabigatran.    
Benzamidine is a competitive inhibitor of serine proteases.
Benzamidine is frequently added to cell lysates, especially yeast cell extracts. It is also a preferred protease inhibitor in protein crystallography. p-Aminobenzamidine immobilized on agarose is used in the isolation of serine proteases by affinity chromatography.
 
Bestatin
https://en.wikipedia.org/wiki/Ubenimex
Target Enzymes: Aminopeptidases
Ubenimex (INN), also known more commonly as bestatin, is a competitive, reversible protease inhibitor. It is an inhibitor of arginyl aminopeptidase (aminopeptidase B),[2] leukotriene A4 hydrolase(a zinc metalloprotease that displays both epoxide hydrolase and aminopeptidase activities),[3] alanyl aminopeptidase (aminopeptidase M/N),[4] leucyl/cystinyl aminopeptidase(oxytocinase/vasopressinase),[5][6] and membrane dipeptidase (leukotriene D4 hydrolase).
It is being studied for use in the treatment of 
acute myelocytic leukemia[7] and lymphedema[8]. It is derived from Streptomyces olivoreticuli.[9] Ubenimex has been found to inhibit the enzymatic degradation of oxytocinvasopressinenkephalins, and various other peptides and compounds.[citation needed]
Chymostatin
 
https://www.biosynth.com/products/products/C-5981.html
Target Enzymes: Serine Proteases,
Cysteine Proteases, Papain.
Strongly inhibit proteases such as chymotrypsin and analogous serine proteinases, papain, chymases, and cathepsins A,B,C, B, H, and L etc that belong to lysosomal cysteine proteinases catagory and the soluble Ca2+ activated proteinase.
Compositions of Chymostatin with other protease inhibitors is often applied for use with plant extracts, especially for young plant tissues which may frequently express serine protease with sensitivity to chomostatin.
Chomostatin has weak inhibitions for human leucocyte elastase
 
E-64
 https://www.biosynth.com/products/products/E-0010.html
 Target Enzymes:
Cysteine Proteases
 (Papain, Cathepsin)
E-64 irreversibly inhibit cysteine proteases (calpain, papain, and cathepsin B, cathepsin L) with high selectivity and potency, acting by forming a thioether bond with the thiol functional group of the active cysteine, and does not affect the functional thiol group of cysteine residues in non-protease enzymes, as creatine kinase or L-lactate dehydrogenase.
Trypsin is the only one serine proteases that E-64 inhibits.
E-64 have capability to restore HIV defective immune responses and inhibition of activation-induced programmed cell death.
E-64 has application for cysteine proteases affinity purification, where it is coupled to a thiolated affinity matrix as an effective ligand.
The coupled E-64 no longer binds substrates irreversibly yet with specificity retained.  
 
EDTA
 https://en.wikipedia.org/wiki/EDTA
 Target Enzymes:
Metalloproteases
 
 
EGTA
 https://en.wikipedia.org/wiki/EGTA_(chemical)
 Target Enzymes:
Ca(II)-Proteases
EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid), also known as egtazic acid (INNUSAN),[1] is an aminopolycarboxylic acid, a chelating agent. It is a colourless solid that is related to the better known EDTA.
Compared to EDTA, it has a lower affinity for 
magnesium, making it more selective for calciumions. It is useful in buffer solutions that resemble the environment in living cells[2] where calcium ions are usually at least a thousandfold less concentrated than magnesium.
EGTA has also been used experimentally for the treatment of animals with cerium poisoning and for the separation of thorium from the mineral monazite.
 EGTA is used as a compound in elution buffer in the Protein Purification technique known as
Tandem Affinity Purification, in which recombinant fusion proteins are bound to calmodulin beads and eluted out by adding EGTA.
EGTA is often employed in dentistry and endodontics for the removal of the smear layer.
 
Leupeptin
 https://en.wikipedia.org/wiki/Leupeptin
 Target Enzymes:
Serine and
Cysteine Proteases
.Leupeptin, also known as N-acetyl-L-leucyl-L-leucyl-L-argininal, is a naturally occurring protease inhibitor that can inhibit cysteine, serine and threonine peptidases.
It is often used during 
in vitro experiments when a specific enzymatic reaction is being studied. When cells are lysed for these studies, proteases, many of which are contained within lysosomes, are released.
These proteases, if freely present in the lysate, would destroy any products from the reaction being studied, and make the experiment uninterpretable.
For example, leupeptin could be used in a 
calpain extraction to keep calpain from being hydrolyzed by specific proteases. The suggested concentration is 1-10 µM (0.5-5 µg/ml).
Leupeptin is an organic compound produced by 
actinomycetes, which inhibits serinecysteine and threonine proteases.
Leupeptin inhibits serine proteinases (
trypsin (K
i=3.5 nM), plasmin (Ki= 3.4 nM), porcine kallikrein), and cysteine proteinases (papaincathepsin B (Ki = 4.1 nM), endoproteinase Lys-C).
It does not inhibit α-
chymotrypsin or thrombin.
Leupeptin is a competitive transition state inhibitor and its inhibition may be relieved by an excess of substrate.

Leupeptin is soluble in water (stable for 1 week at 4 °C and 1 month at −20 °C), ethanol, acetic acid and DMF.
It can be given topically for middle and inner ear infections.
[1]
 
Pepstatin A
 https://en.wikipedia.org/wiki/Pepstatin
https://en.wikipedia.org/wiki/Statine

 Target Enzymes:
Aspartic Proteases (eg. HIV)
Pepstatin is a potent inhibitor of aspartyl proteases.
It is a hexa-
peptide containing the unusual amino acid statine (Sta, (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid), having the sequence Isovaleryl-Val-Val-Sta-Ala-Sta (Iva-Val-Val-Sta-Ala-Sta).[1] It was originally isolated from cultures of various species of Actinomyces[1] due to its ability to inhibit pepsin at picomolar concentrations.[2]
Pepstatin A is well known to be an inhibitor of aspartic proteinases such as pepsin, cathepsins D and E. 
 
Phenylmethylsulfonyl fluoride (PMSF)
 https://en.wikipedia.org/wiki/PMSF
 Target Enzymes: Serine Proteases
.In biochemistryphenylmethane sulfonyl fluoride or phenylmethylsulfonyl fluoride (PMSF) is a serine protease inhibitorcommonly used in the preparation of cell lysates. PMSF does not inhibit all serine proteases[citation needed]. It is rapidly degraded in water and stock solutions are usually made up in anhydrous ethanolisopropanolcorn oil, or DMSO.
PMSF binds specifically to the active site 
serine residue in a serine protease. It does not bind to any other serine residues in the protein. 
The median lethal dose is less than 500 mg/kg[citation needed].
PMSF is a cytotoxic chemical which should be handled only inside a fume hood.

PMSF is commonly used in protein solublization in order to deactivate proteases from digesting proteins of interest after cell lysis.
 
Reduction Agents in Downstream processing
                                                                                
The disulfide bonds are extremely rare in cytosolic proteins, since the cytosol (intracellular fluid) is generally a reducing environment.
https://en.wikipedia.org/wiki/Protein_structure#Tertiary_structure
https://en.wikipedia.org/wiki/Disulfide#Occurrence_in_proteins
Disulfide Reduction Reagents reduce oxidation of a protein sample, inhibit the oxidation of free sulfhydryl residues, maintaining SH groups in the reduced state, and its ability thereby, preserve enzymatic activity.
Its usefulness also stems from its water solubility, reduced odor, and lower toxicity.
 
Tris-(2-carboxyethyl)phosphine HCl (TCEP)
https://en.wikipedia.org/wiki/TCEP
TCEP (tris(2-carboxyethyl)phosphine) is a reducing agent frequently used in biochemistry and molecular biology applications.[1]
It is often prepared and used as a hydrochloride salt (TCEP-HCl) with a molecular weight of 286.65 gram/mol.
It is soluble in water and available as a stabilized solution at neutral pH and immobilized onto an agarose support to facilitate removal of the reducing agent.
TCEP is often used as a reducing agent to break disulfide bonds within and between proteins as a preparatory step for gel electrophoresis.
Compared to the other two most common agents used for this purpose (dithiothreitol and β-mercaptoethanol), TCEP has the advantages of being odorless, a more powerful reducing agent, an irreversible reducing agent (in the sense that TCEP does not regenerate—the end product of TCEP-mediated disulfide cleavage is in fact two free thiols/cysteines), more hydrophilic, and more resistant to oxidation in air.[2]
It also does not reduce metals used in immobilized metal affinity chromatography.
TCEP is particularly useful when labeling cysteine residues with maleimides. TCEP can keep the cysteines from forming di-sulfide bonds and unlike dithiothreitol and β-mercaptoethanol, it will not react as readily with the maleimide.[2] However, TCEP has been reported to react with maleimide under certain conditions.[3][4]
TCEP is also used in the tissue homogenization process for RNA isolation.[5]
.
 
1,4-Dithioerythritol (DTE)
https://en.wikipedia.org/wiki/Dithioerythritol
Dithioerythritol (DTE) is a sulfur containing sugar derived from the corresponding 4-carbon monosaccharide erythrose. It is an epimer of dithiothreitol (DTT). The molecular formula for DTE is C4H10O2S2.
Like DTT, DTE makes an excellent reducing agent, although its standard reduction potential is not quite as negative, i.e., DTE is slightly less effective at reducing than DTT. This is presumably because the orientation of the OH groups in its cyclic disulfide-bonded form (oxidized form) is less stable due to greater steric repulsion than their orientation in the disulfide-bonded form of DTT. In the disulfide-bonded form of DTT, these hydroxyl groups are trans to each other, whereas they are cis to each other in DTE.
 
1,4-Dithiothreitol (Cleland's Reagent, DTT)
https://en.wikipedia.org/wiki/Dithiothreitol
Dithiothreitol (DTT) is the common name for a small-molecule redox reagent also known as Cleland's reagent.[2]
DTT has an 
epimeric('sister') compound, dithioerythritol (DTE).
DTT is used as a reducing or "deprotecting" agent for thiolated DNA.
The terminal sulfur atoms of thiolated 
DNA have a tendency to form dimers in solution, especially in the presence of oxygen. Dimerization greatly lowers the efficiency of subsequent coupling reactions such as DNA immobilization on gold in biosensors. 
The DTT removal procedure is often called "desalting."
Generally, DTT is used as a protecting agent that prevents oxidation of 
thiol groups.
DTT is frequently used to reduce the 
disulfide bonds of proteins and, more generally, to prevent intramolecular and intermolecular disulfide bonds from forming between cysteine residues of proteins.
However, even DTT cannot reduce buried (solvent-inaccessible) disulfide bonds, so reduction of disulfide bonds is sometimes carried out under 
denaturing conditions (e.g., at high temperatures, or in the presence of a strong denaturant such as 6 M guanidinium chloride, 8 M urea, or 1% sodium dodecylsulfate).
 
2-Mercaptoethanol
https://en.wikipedia.org/wiki/2-Mercaptoethanol
2-Mercaptoethanol (also β-mercaptoethanolBME2BME2-ME or β-met) is the chemical compound with the formula HOCH2CH2SH. ME or βME, as it is commonly abbreviated, is used to reduce disulfide bonds and can act as a biological antioxidant by scavenging hydroxyl radicals (amongst others). It is widely used because the hydroxyl group confers solubility in water and lowers the volatility. Due to its diminished vapor pressure, its odor, while unpleasant, is less objectionable than related thiols.
Applications: Reducing proteins, Preventing protein oxidation, Denaturing ribonucleases.
 


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