Enzyme Substrates

Horseradish peroxidase is a common marker enzyme for antibodies, and can catalyze the chemiluminescent oxidation of luminol in the presence of hydrogen peroxide. This transformation is used in ultra-sensitive ELISA assays. Luminescence can be detected at very low levels using extremely sensitive scintillation counters.

Luminol is also used for the detection of blood stains in criminal investigation. For this, luminol is mixed with a liquid containing hydrogen peroxide. If the mixture comes in contact with hemoglobin, the iron in the hemoglobin accelerates chemiluminescent oxidation of luminol.

Magenta-Gal is used as an alternative to X-Gal, and produces a red color. Magenta-Gal is hydrolyzed by β-galactosidase to produce a red precipitate. This precipitate is insoluble in alcohol and xylenes, thus making it suitable for immunoblotting and immunocytochemical assays. Bacterial colonies containing active β-galactosidase produce red colonies when grown on plates containing Magenta-Gal.

Magenta-Gal is commonly used in conjunction with IPTG (GoldBIo #I2481) for red/white screening.

Magenta-phosphate p toluidine salt; 5-Bromo-6-chloro-3-indoxyl phosphate, p-toluidine salt

Mildronate is a pharmaceutical amino acid amide believed to stimulate vasodilation, thus used to treat angina and myocardial infraction. Chemically, it identifies with aromatic ethers and secondary alcohols. It is a substrate for γ-butyrobetaine dioxygenase and structural analogue of γ-butyrobetaine, competitively inhibiting by binding to the γ-butyrobetaine hydroxylase substrate.

It is a biological regulator to alpha 2-adrenergic receptors as well as nitric oxide-dependent mechanisms, functions which reduce blood glucose concentrations through oxidation. In ocular and cerebral ischemia, Mildronate decreases symptoms of circulatory disturbance through inhibition of carnitine synthesis.

4-Methylumbelliferyl beta-D-glucuronide (MUG) is a fluorogenic substrate for beta-glucuronidase (GUS). Upon cleavage, MUG yields blue fluorescence seen under UV light.

This substrate is commonly used for the detection of Escherichia coli through its ability to detect activity of beta-glucuronidase, which is encoded by uidA. GUS activity is tested in minimal medium o-nitrophenyl-beta-D-galactopyranoside (MMO)-MUG.

It is ideal for plant molecular study as a way to examine gene expression under various promoters. And while it works well for measuring activity in plant lysate and leaf disks, it can also work in whole plant assays.

N-Acetyl-L-carnosine, or N-Acetylcarnosine (abbreviated NAC) is a dipeptide. It is similar to carnosine but more resistant to carnosinase degradation thanks to the addition of an acetyl group.

Chromogenic substrate for beta-D-galactosidase, yielding a green precipitate upon cleavage

N-Succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-nitroanilide is a colorimetric substrate for peptidyl prolyl isomerase, chymotrypsin and human leukocyte cathepsin G.

Nitrotetrazolium Blue (NBT) is used in combination with 5-bromo-4-chloro-3-indoxyl phosphate (BCIP) to detect alkaline phosphatase activity in blotting techniques and in situ hybridization chemistry. BCIP acts as the substrate for alkaline phosphatase, and the oxidizing reagent NBT enhances the purple-brown final color of the precipitates at the reaction sites.

NBT has also been used with X-Gal,or Salmon-Gal to speed up and enhance colony screening.

Phenyl-β-D-galactopyranoside is a noninducing β-galactosidase substrate. It is used in selection of mutants of lacZ plasmid-based transgenic mice. It is an acceptor substrate in fucosyltransferase detection.

PNPG (Gus) is a chromogenic β-glucuronidase substrate, yielding a yellow solution upon cleavage. It is used to detect E. coli contamination in food and drinking water due to their β-glucuronidase activity.

Nonfluorescent resazurin, which is reduced by viable cells to red-fluorescent resorufin, can be used to detect the metabolic activity of many cell types from bacteria to higher eukaryotes and is a sensitive fluorogenic substrate for fluorometric and UV-spectrophotometric assays of phosphatases. Resazurin is used in the Resazurin Microtiter Assay for rapid detection of multidrug-resistant Mycobacterium tuberculosis, in cell viability tests, and to assay mitochondrial functions in vitro.