Enzyme Substrates

BCIP Sodium salt is a water soluble form of BCIP. BCIP is a chromogenic substrate that is used to visualize the activity of alkaline phosphatase. When combined with NBT, BCIP produces a blue to violet insoluble end product and can be used in a variety of assays, including immunohistochemistry, Western blotting, Northern blotting and Southern blotting.

1-O-Methyl-β-D-glucuronic acid, sodium salt is an inducer of β-glucuronidase, a glycosidase enzyme coded by the GUS gene.

The GUS gene is the most extensively used reporter gene, particularly in plant biology because it offers the advantage of having no background activity.

β-glucuronidase also has an important role within human digestion because this enzyme is capable of deconjugating toxins within the body and releasing them back freely into the bloodstream. There is a positive relationship between higher levels of β-glucuronidase in the colon and increased cancer risk. However, β-glucuronidase, at certain levels, is necessary, and deficiencies can result in mucopolysaccharidosis type VII or Sly syndrome in newborns.

Tetrazolium red is used to visualize dehydrogenase enzyme activity. Initially, the tetrazolium solution is colorless but changes to red when it comes into contact with hydrogen. Tetrazolium red is used in a biochemical viability test for seeds. The test relies on dehydrogenase enzymes to release hydrogen ions, which subsequently reduce the colorless tetrazolium salt solution to a red compound called formazan. Living cells turn red while dead cells remain colorless.

2-Naphthyl caprylate is a chromogenic substrate for carboxylesterase and lipase. 2-naphthol is released upon hydrolyzation. By simultaneous coupling with a diazonium salt, the corresponding azo-dye is formed.

Naphthols can also be detected by fluorescence analysis, and can be used in a colorimetric assay for the extracellular lipase of Pseudomonas fluorescent B52.

2-Naphthyl-beta-D-galactopyranoside is a substrate for beta-galactosidase. 2-naphthol is released upon hydrolyzation. By simultaneous coupling with a suitable staining reagent, such as hexazonium p-rosaniline, the corresponding red-brown azo-dye is formed. Naphthols can also be detected by fluorescence analysis.

2-Naphthyl-α- L-fucopyranoside is a chromogenic substrate for α-L-Fucosidase. 2-naphthol is released upon hydrolyzation. By simultaneous coupling with a suitable staining reagent, such as hexazonium p-rosaniline, the corresponding azo-dye is formed. Naphthols can also be detected by fluorescence analysis.

2-Naphthyl-α-D-glucopyranoside is a substrate for α-glucosidase. 2-naphthol is released upon hydrolyzation. By simultaneous coupling with a suitable staining reagent, such as hexazonium p-rosaniline, the corresponding red-brown azo-dye is formed. Naphthols can also be detected by fluorescence analysis.

2-Nitrophenyl-1-thio-β-D-galactopyranoside is an enzyme substrate that is often used in the detection of lac- mutations.

2-Nitrophenyl-N-acetyl- β-D- glucosaminide is a chromogenic β-glucosaminidase substrate yielding a yellow solution upon cleavage.

3-Indoxyl phosphate bis (2-amino-2-methyl- 1,3-propanediol) salt
Y™-phosphate bis(2-amino-2-methyl-1,3-propanediol) salt
Y™-phos • 2 AMPD

P-aminophenyl phosphate (pAPP) can be used as a substrate for alkaline phosphatase enzymatic reactions in order to electrochemically quantify ALP activity.

The enzymatic reaction results in the production of phenol, which can be quantified and used to analyze ALP concentrations.

ALP is a naturally occurring enzyme and can be found in human tissue in the liver, bone and intestines. Usual concentrations of ALP range between 25-100 IU/L. A significant spike outside of that range could act as a signal for many diseases.

4-Aminophenyl-β-D-galactopyranoside is a substrate for β-galactosidase. It is also a weak β-D-galactosidase inducer and can be used in affinity chromatography for isolation of galactose-binding. The 4-aminophenyl releases upon cleavage by β-galactosidase. It can then be assayed by electro-oxidation to 4-imino quinone while recording changes in the potential or current galvanic system.

Substrate for horseradish peroxidase (HRP). HRP catalyzes oxidation of 4-chloro-1-naphthol in the presence of peroxide to the blue, insoluble product 4-chloro-1-naphthon. It is used for both blotting and immunohistochemical staining, but is less sensitive than other precipitating HRP substrates.

4-Methylumbelliferyl caprylate is a fluorogenic substrate for esterase with C8 activity, yielding a blue fluorescent solution upon cleavage and is used for the identification of Salmonella spp. based on the detection of caprylate esterase.

4-Methylumbelliferyl palmitate is used as a fluorogenic substrate for a subclass of esterases called lipase.

Lipase is an enzyme that assists in the hydrolysis of fats. They have a large role in digestion as well as dietary lipid transport.

Lysosomal acid lipase (LAL), in particular, is required for the hydrolysis of cholesterol esters which are then delivered into the lysosome. Deficiencies in LAL are primarily associated with two diseases: cholesteryl ester storage disease (CESD) and Wolman’s disease (WD). 4-Methylumbelliferyl palmitate has often been used as one of the lipase substrates in studies involving either of these diseases.

Sensitive fluorogenic substrate for fluorometric and UV-spectrophotometric assays of phosphatases.

4-Methylumbelliferyl phosphate (MUP) is a sensitive fluorogenic substrate for fluorometric and UV-spectrophotometric assays of phosphatase.

Phosphotase activity has long been studied due to its importance in cellular function. Upon dephosphorylation, MUP yields fluorescent 4-methylumelliferone (4-MU). This reaction has been used to spot PCR amplified products in ELISA and to classify bacteria.

More specifically, MUP can act as an indicator of C. perfringens because of its ability to detect phosphatase which is a highly specific indicator of C. perfringens.