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Proteases

Proteases, also known as peptidases, are a large class of enzymes that play key roles in protein activation, cell regulation and signaling, as well as in the generation of amino acids for protein synthesis or utilization in other metabolic pathways. Proteases primarily act as catalysts in proteolysis -the hydrolysis of the peptide bonds within proteins - breaking down complex proteins into either shorter polypeptide fragments or single amino acids. Ultimately, protease activity can activate a function, promote a signaling pathway or result in a destructive change (e.g. abolishing a protein's function or digesting it to its principal components). Because of its significance, monitoring various protease activities have become a routine task for many biological laboratories, and more importantly proteases are considered promising drug targets for an onslaught of diseases. To assess enzymatic activities or screen for inhibitors of known and various proteases, such as serine proteinases, aspartic proteinases, or matrix metalloproteinases (MMPs), browse our wide-selection of fluorescence-based assays and known protease substrates.

 

 

Major Protease Classes


Proteases are divided into four major groups according to the character of their catalytic active site and conditions of action. These groups include: serine proteinases, cysteine (thiol) proteinases, aspartic proteinases, or matrix metalloproteinases (MMPs). Attachment of a protease to a certain group depends on the structure of catalytic site and the amino acid (as one of the constituents) essential for its activity. Some can detach the terminal amino acids from the protein chain (exopeptidases), while others attack internal peptide bonds of a protein (endopeptidases).

Metabolic Proteases


Proteases are used throughout an organism for various metabolic processes. Acid proteases (e.g. pepsin) secreted into the stomach and serine proteases (e.g. trypsin and chymotrypsin) present in duodenum enable us to digest the protein in food. Proteases present in blood serum (e.g. thrombin, plasmin, Hageman factor, etc.) play important roles in blood-clotting, as well as blood clot lysis, and the correct action of the immune system. Other proteases are present in leukocytes (e.g. elastase and cathepsin G) and play several different roles in metabolic control.

Protease Biodiversity


Bacteria also secrete proteases to hydrolyze (digest) the peptide bonds in proteins and therefore break the proteins down into their constituent monomers (amino acids). Bacterial and fungal proteases are particularly important to the global carbon and nitrogen cycles in the recycling of proteins, and such activity tends to be regulated by nutritional signals in these organisms. The net impact of nutritional regulation of protease activity among the thousands of species present in soil can be observed at the overall microbial community level as proteins are broken down in response to carbon, nitrogen, or sulfur limitation. A secreted bacterial protease may also act as an exotoxin, and be an example of a virulence factor in bacterial pathogenesis. Bacterial exotoxic proteases destroy extracellular structures.

 

Detecting Protease Activity



Trypsin protease activity was analyzed by Amplite® Universal Fluorimetric Protease Activity Assay Kit. Protease substrate was incubated with 1 unit trypsin in the kit assay buffer. The control wells had protease substrate only (without trypsin). The fluorescence signal was measured starting from time 0 when trypsin was added. Samples were done in triplicates.
The Amplite® Universal Fluorimetric Protease Activity Assay is a proven tool for performing routine protease isolation assays or for identifying the presence of contaminating proteases in protein samples. This homogenous fluorescence quenching based assay utilizes a univeral protease substrate that can accurately quantitate a broad range of protease activities including trypsin, chymotrypsin, thermolysin, proteinase K, protease XIV, and elastase. In its entirety the univeral peptidase/protease substrate, which is heavily labeled with multiple fluorophores, is non-fluorescent due to significant collisional self-quenching. Upon protease-catalyzed hydrolysis, this quenching effect is eliminated, yielding brightly fluorescent dye-labeled short peptides. The magnitude of the fluorescence signal is proportional to the extent of substrate cleavage and can be used to quantitate protease activity. The assay can be performed in a convenient 96-well or 384-well microtiter plate format and readily adapted to automation.

Also available are casein-FITC and casein-TAMRA conjugates for quantitating protease activity. Casein is considered to be a generic substrate for a broad spectrum of proteases.
 

Table 1. Fluorimetric assay kits for measuring the activity of various proteases.

Kit
Ex (nm)
Em (nm)
Cutoff (nm)
Unit Size
Cat No.
Amplite® Universal Fluorimetric Protease Activity Assay Kit *Green Fluorescence*490 nm525 nm515 nm500 tests13500
Amplite® Universal Fluorimetric Protease Activity Assay Kit *Red Fluorescence*540 nm590 nm570 nm500 tests13501

Table 2. Generic substrates for measuring the activity of various proteases.

Name
Target
Ex (nm)
Em (nm)
ε¹
Unit Size
Cat No.
Casein, FITC-conjugatedProtease substrate491 nm516 nm73,0005 mg13440
Casein, FITC-conjugatedProtease substrate552 nm578 nm90,0005 mg13441
  1. ε: molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).

 

Matrix Metalloproteinases (MMPs) Assays



MMP activity monitored using Amplite® Universal Fluorimetric MMP Activity Assay. APMA-activated MMPs (30 ng each) were mixed with MMP Green™ substrate. One hour after starting the reaction the fluorescence signal was monitored using a NOVOStar microplate reader (BMG Labtech) at Ex/Em = 490/525 nm. The reading from all wells was subtracted by the reading from substrate control, which contains MMP Green™ substrate but no MMPs. The MMP Green™ substrate can detect the activity of sub-nanogram of all MMPs (n=3).
Matrix metalloproteinases (MMPs) are a family of calcium-dependent zinc-containing endopeptidases. Collectively, these enzymes are responsible for the degradation of most extracellular matrix proteins during growth and normal tissue turnover (e.g. breakdown of connective tissues, bone remodeling, the menstrual cycle and repair of tissue damage). MMPs are also key regulators of cell behavior and signaling pathways such as cell proliferation, migration, differentiation and apoptosis. They are known to be involved in the cleavage of cell surface receptors, the release of apoptotic ligands, and cytokine inactivation. While the exact contribution of MMPs to certain pathological processes is difficult to assess, MMPs appear to play a key role in the development of arthritis as well as in the invasion and metastasis of cancer.

Detecting Matrix Metalloproteinase Activity


The Amplite® Universal Fluorimetric MMP Activity Assay kits offer a convenient FRET-based method for the accurate quantitation of a wide range of matrix metalloproteinase activities. The Amplite® MMP substrates are comprised of a fluorophore and a dark quencher separated by an amino acid sequence. In this confirmation, the substrates are non-fluoresecent. Upon MMP-catalyzed hydrolysis, this quenching effect is eliminated, yielding brightly fluorescent dye-labeled short peptides. The magnitude of the fluorescence signal is proportional to the extent of substrate cleavage and can be used to quantitate MMP activity. The assay can be performed in a convenient 96-well or 384-well microtiter plate format, and if using purified MMP enzymes the Amplite® Universal Fluorimetric MMP Activity Assay kits can successfully screen for MMP inhibitors. The Amplite® MMP Green™ and MMP Red™ substrates used in these assays are also available as separately as Cat No. 13520 and 13521, respectively.
 

Table 3. Fluorimetric assay kits for measuring the activity of various matrix metalloproteinases.

Kit
Ex (nm)
Em (nm)
Cutoff (nm)
Unit Size
Cat No.
Amplite® Universal Fluorimetric MMP Activity Assay Kit *Green Fluorescence*490 nm525 nm515 nm100 tests13510
Amplite® Universal Fluorimetric MMP Activity Assay Kit *Red Fluorescence*540 nm590 nm570 nm100 tests13511

Table 4. Generic substrates for measuring the activity of various matrix metalloproteinases.

Name
Ex (nm)
Em (nm)
Unit Size
Cat No.
MMP Green™ substrate494 nm515 nm100 tests13519
MMP Green™ substrate494 nm515 nm1 mg13520
MMP Red™ substrate545 nm572 nm100 tests13522
MMP Red™ substrate545 nm572 nm1 mg13521

 

Matrix Metalloproteinase-3 (MMP-3) Assays


Matrix Metalloproteinase-3 (MMP-3) also known as stromelysin-1, constitutes a family of zinc-dependent endopeptidases that function within the extracellular matrix. These enzymes are responsible for the breakdown of connective tissues and are important in bone remodeling, the menstrual cycle, and repair of tissue damage. In addition to breaking down numerous extracellular matrix components, MMP-3 can activate other MMPs (such as MMP-1, MMP-7 and MMP-9), gelatinase B, the collagenases and several serpin-type serine proteinase inhibitors. Moreover, it can release a number of cell surface molecules such as E-cadherin, L-selectin, heparin-binding EFG-like growh factor, and TNF-?, which can exert oncogenic effects.

Detecting MMP-3 Activity



Dose response of MMP-3 enzyme activity was measured with Amplite® MMP-3 Activity Assay Kit using a NOVOStar microplate reader (BMG Labtech).
Amplite® Fluorimetric MMP-3 Activity Assay Kit provides a quick and sensitive method to measure MMP-3 enzyme activity, as well as, screen for MMP-3 inhibitors. This assay utilizes a fluorescence resonance energy transfer (FRET) peptide sequence that is highly selective for MMP-3 hydrolysis over other MMP enzymes. In the intact FRET peptide, the fluorescence of Tide Fluor™ 2 (TF2) is quenched by Tide Quencher™ 2. After cleavage into two separate fragments by MMP-3 hydrolysis, the green fluorescence of TF2 is recovered. With excellent fluorescence quantum yield and longer wavelength, TF2 shows less interference from autofluorescence of test compounds and cellular components, and is much more sensitive than an EDANS/Dabcyl FRET substrate. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format. The perfect excitation of TF2 at 488 nm makes the assay readily readable with almost all the common fluorescence instruments equipped with argon laser and FITC filter set.
 

Table 5. Fluorimetric assay kits for measuring the activity of matrix metalloproteinase-3.

Kit
Ex (nm)
Em (nm)
Cutoff (nm)
Unit Size
Cat No.
Amplite® MMP-3 Activity Assay Kit *Green Fluorescence*490 nm525 nm515 nm100 tests13512

Table 6. Generic substrates for measuring the activity of matrix metalloproteinase-3.

Name
Ex (nm)
Em (nm)
Unit Size
Cat No.
MMP-3 Green™ substrate494 nm515 nm100 tests13527
MMP-3 Green™ substrate494 nm515 nm1 mg13528

 

Proteasome 20S


Proteasomes are protein complexes inside all eukaryotes, archaea, and in some bacteria. In eukaryotes, proteasomes are located in the nucleus and cytoplasm. The main function of the proteasome is to degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds. Enzymes that carry out such reactions are called proteases.

Function


Proteasomes are part of a major mechanism by which cells regulate the concentration of particular proteins and degrade misfolded proteins. The degradation process yields peptides of about seven to eight amino acids long, which can then be further degraded into shorter amino acid sequences and used in synthesizing new proteins. Proteins are tagged for degradation with a small protein called ubiquitin. The tagging reaction is catalyzed by enzymes called ubiquitin ligases. Once a protein is tagged with a single ubiquitin molecule, this is a signal to other ligases to attach additional ubiquitin molecules. The result is a polyubiquitin chain that is bound by the proteasome, allowing it to degrade the tagged protein.

Structure


In structure, the proteasome is a cylindrical complex containing a "core" of four stacked rings forming a central pore. Each ring is composed of seven individual proteins. The inner two rings are made of seven B subunits that contain three to seven protease active sites. These sites are located on the interior surface of the rings, so that the target protein must enter the central pore before it is degraded. The outer two rings each contain seven ? subunits whose function is to maintain a "gate" through which proteins enter the barrel. These ? subunits are controlled by binding to "cap" structures or regulatory particles that recognize polyubiquitin tags attached to protein substrates and initiate the degradation process. The overall system of ubiquitination and proteasomal degradation is known as the ubiquitin-proteasome system.

Measuring Proteasome 20S Activity



Detection of proteasome activity in Jurkat cells with Amplite® Fluorimetric Proteasome 20S Activity Assay Kit. Jurkat cells were seeded on the same day at 500,000 cells/90 µL/well in a 96-well black wall/clear bottom Costar plate. The cells were treated with or without 50 mM H2O2 for 30 minutes. The proteasome assay loading solution (100 µL/well) was added and incubated in a 5% CO2, 37°C incubator for 3 hours. The fluorescence intensity was measured at Ex/Em = 490/525 nm using a Gemini fluorescent microplate reader (Molecular Devices).
The most common form of the proteasome in this pathway is the 26S proteasome, an ATP-dependent proteolytic complex, which contains one 20S (700-kDa) core particle structure and two 19S (700-kDa) regulatory caps. The 20S core contains three major proteolytic activities including chymotrypsin-like, trypsin-like and caspase-like activities. It is responsible for the breakdown of the key proteins involved with apoptosis, DNA repair, endocytosis, and cell cycle control.

Amplite® Fluorimetric Proteasome 20S Activity Assay Kit is a homogeneous assay that measures the chymotrypsin-like protease activity associated with the proteasome complex in cultured cells. This kit uses LLVY-R110 as a fluorogenic indicator for proteasome activities. Cleavage of LLVY-R110 by proteasome generates strongly green fluorescent R110 which can be easily monitored by a fluorescence microplate reader at Ex/Em = 490/525 nm. The assay is robust and can be readily adapted for high-throughput assays to evaluate the proteasome activities or screen the inhibitors in cultured cells or in solution. The assay can be performed in a convenient 96-well and 384-well fluorescence microtiter-plate format.
 

Table 7. Fluorimetric assay kits for measuring the activity of proteasome 20S.

Kit
Ex (nm)
Em (nm)
Cutoff (nm)
Unit Size
Cat No.
Amplite® Fluorimetric Proteasome 20S Activity Assay Kit *Green Fluorescence*490 nm525 nm515 nm100 tests13456

Table 8. Generic substrates for measuring the activity of proteasome 20S.

Name
Target
Ex (nm)
Em (nm)
ε¹
Unit Size
Cat No.
(Ac-ANW)2R110Proteasome 20S-Β5i500 nm522 nm800001 mg13455
(Ac-KQL)2R110Proteasome 20S-Β2i500 nm522 nm800001 mg13465
(Ac-PAL)2R110Proteasome 20S-Β1i500 nm522 nm800001 mg13467
(Ac-WLA)2R110Proteasome 20S-Β5c500 nm522 nm800001 mg13468
Suc-LLVY-AMCProteasome 20S-Β5c341 nm441 nm190001 mg13453
Suc-LLVY-AminoluciferinProteasome 20S-Β5c362 nm499 nm180001 mg13452
(Suc-LLVY)2R110Proteasome 20S-Β5c500 nm522 nm800001 mg13451
(Z-LLE)2R110Proteasome 20S-Β1c500 nm522 nm800001 mg13466
  1. ε: molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).

 

Renin Assays



Renin dose responses were measured with Amplite® Fluorimetric Renin Assay Kit on a 96-well black solid plate using a Gemini fluorescence microplate reader (Molecular Devices). As low as 0.01 µg/mL renin was detected with 30 minutes incubation (n=3).
Renin is an enzyme that participates in the body's renin-angiotensin system (RAS) that mediates extracellular volume and arterial vasoconstriction. It regulates blood pressure and electrolyte homoeostasis. Angiotensin II constricts blood vessels leading to increased blood pressure. It also increases the secretion of ADH and aldosterone, and stimulates the hypothalamus to activate the thirst reflex. An over-active renin-angiotension system leads to vasoconstriction and retention of sodium and water. Renin has been identified to be an attractive target for the treatment of hypertension.

Measuring Renin Activity


Amplite® Fluorimetric Renin Assay Kit provides a quick and convenient assay for high throughput screening of renin inhibitors and renin activity. The assay utilizes a fluorescence resonance energy transfer (FRET) peptide modified with the FRET pair Tide Fluor™ 3 (TF3) and Tide Quencher™ 3 (TQ3). In the intact FRET peptide, the fluorescence of TF3 is quenched by TQ3. Upon cleavage into two separate fragments by renin, the fluorescence of TF3 is recovered, and the fluorescence signal can be easily monitored by a fluorescence microplate reader at Ex/Em = 540/590 nm. This assay is approximately fifty fold more sensitive than an EDANS/Dabcyl-based assay, detecting as little as 1 ng renin in a 100 µL reaction volume.
 

Table 9. Fluorimetric assay kits for measuring the activity of renin.

Kit
Ex (nm)
Em (nm)
Cutoff (nm)
Unit Size
Cat No.
Amplite® Fluorimetric Renin Assay Kit *Red Fluorescence*540 nm590 nm570 nm100 tests13530

 

Peptidase and Protease Substrates


In general, peptidases cleave shorter peptides, and proteases cleave longer peptides and proteins. Depending on their sites of cleavage, peptidases can be classified as exopeptidases if they preferentially hydrolyze amino acid residues from the terminus of a peptide, or endopeptidases if they cleave internal peptide bonds. Exopeptidases are further divided into aminopeptidases and carboxypeptidases depending on whether they hydrolyze residues from the amine or the carboxy terminus.

Although the spectral properties of fluorogenic peptidase substrates and their hydrolysis products are easily predictable, the utility of a given substrate for an enzyme depends on the kinetics of hydrolysis by the enzyme, which, in turn, depends on the substrate's concentration and amino acid sequence, as well as on the pH, temperature and presence of cofactors in the medium. Two common fluorescent dyes for labeling peptides include the blue fluorescent dye 7-Amino-4-methylcoumarin (AMC) and the red fluorescent dye Rhodamine 110 (R110). Both peptide-modified AMC and R110 substrates have been used extensively for detecting peptidase activity in cell, homogenates and solutions.
 

Table 10. Substrates for measuring protease activity in cells, homogenates or solution.

Substrate
Target
Ex (nm)
Em (nm)
ε¹ (nm)
Unit Size
Cat No.
Ala-AMCAlanyl aminopeptidase and trypsin341 nm441 nm190005 mg13458
BOC-Val-Pro-Arg-AMCAlpha-thrombin341 nm441 nm190005 mg13461
(BOC-VPR)2R110Alpha-thrombin500 nm522 nm800001 mg13462
D-Ala-AMC *CAS 201847-52-1*Aminopeptidase and endopeptidase341 nm441 nm190005 mg13459
D-VLK-AMC [D-Val-Leu-Lys-AMC]Plasmin341 nm441 nm190005 mg13201
Gly-Pro-AMC *CAS: 115035-46-6*Dipeptidyl peptidase IV (DPPIV)341 nm441 nm190005 mg13450
MeO-Succ-Arg-Pro-Tyr-AMCChymotrypsin-like proteases341 nm441 nm190005 mg13460
Pro-AMCTricorn Protease (TRI)341 nm441 nm190005 mg13457
Benzoyl-Arg-AMCHuman alpha-thrombin341 nm441 nm190005 mg13478
Z-Gly-Pro-AMC *CAS 68542-93-8*Post-proline cleaving enzyme (prolyl endopeptidase)341 nm441 nm190005 mg13477
  1. ε: molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).

 

Product Ordering Information


 

Table 11. Ordering Info for Generic Protease Probes and Assays Products

Cat#
Product Name
Unit Size
Price
13440Casein, FITC-conjugated5 mg205
13441Casein, TAMRA-conjugated5 mg205
13500Amplite® Universal Fluorimetric Protease Activity Assay Kit *Green Fluorescence*500 Tests205
13501Amplite® Universal Fluorimetric Protease Activity Assay Kit *Red Fluorescence*500 Tests205

Table 12. Ordering Info for MMP Probes and Assays Products

Cat#
Product Name
Unit Size
Price
13510Amplite® Universal Fluorimetric MMP Activity Assay Kit *Green Fluorescence*100 Tests310
13511Amplite® Universal Fluorimetric MMP Activity Assay Kit *Red Fluorescence*100 Tests310
13512Amplite® MMP-3 Activity Assay Kit *Green Fluorescence*100 Tests310
13519MMP Green™ substrate100 Tests205
13520MMP Green™ substrate1 mg730
13521MMP Red™ substrate1 mg520
13522MMP Red™ substrate100 Tests100
13527MMP-3 Green™ substrate100 Tests100
13528MMP-3 Green™ substrate1 mg520

Table 13. Ordering Info for Proteasome 20S Probes and Assays Products

Cat#
Product Name
Unit Size
Price
13451(Suc-LLVY)2R1101 mg100
13452Suc-LLVY-Aminoluciferin1 mg205
13453Suc-LLVY-AMC1 mg100
13455(Ac-ANW)2R1101 mg205
13456Amplite® Fluorimetric Proteasome 20S Activity Assay Kit *Green Fluorescence*100 tests310
13465(Ac-KQL)2R1101 mg100
13466(Z-LLE)2R1101 mg100
13467(Ac-PAL)2R1101 mg100
13468(Ac-WLA)2R1101 mg100

Table 14. Ordering Info for Renin Probes and Assays Products

Cat#
Product Name
Unit Size
Price
13530Amplite® Fluorimetric Renin Assay Kit *Red Fluorescence*100 tests205

Table 15. Ordering Info for Peptidase Probes and Assays Products

Cat#
Product Name
Unit Size
Price
13000Benzoyl-Leu-Gly-Arg-Aminoluciferin1 mg520
13201D-VLK-AMC [D-Val-Leu-Lys-AMC]5 mg100
13450Gly-Pro-AMC *CAS 115035-46-6*5 mg100
13457Pro-AMC5 mg100
13458Ala-AMC5 mg100
13459D-Ala-AMC *CAS 201847-52-1*5 mg100
13460MeO-Succ-Arg-Pro-Tyr-AMC5 mg100
13461BOC-Val-Pro-Arg-AMC5 mg100
13462(BOC-VPR)2R1101 mg100
13477Z-Gly-Pro-AMC *CAS 68542-93-8*5 mg100