Enzyme Substrates

Resorufin-β-D- glucuronic acid is a fluorogenic substrate for β-glucuronidase, yielding resorufin upon cleavage.

Salmon Gal (6-Chloro-3-indolyl-β-D-galactopyranoside) is used with IPTG as an alternative chromogenic substrate capable of detecting LacZ gene encoded ß-galactosidase activity for colony screening in colometric assays.

Unlike colony blue/white screening through X-Gal, nonrecombination results in a salmon color after salmon gal is cleaved by ß-galactosidase through hydrolysis. This contrast may be useful in comparative or secondary screenings.

Incorporating nitroblue tetrazolium salt (NBT) into the salmon gal reactions provides faster results with a clear, purple precipitate than in unaccompanied uses of salmon gal.

TMB is an excellent colorimetric substrate for detection of horseradish peroxidase labeled probes and is used with peroxidase and peroxidase-coupled systems, particularly in ELISA techniques. It produces a soluble end product, which is blue in color and can be read spectrophotometrically. TMB has also been used in the quantitative determination of hemoglobin and in cytochemical staining for peroxidase. TMB is a hepatitis B antigen.

TMB is an excellent colorimetric substrate for detection of horseradish peroxidase labeled probes and is used with peroxidase and peroxidase-coupled systems, particularly in ELISA techniques. It produces a soluble end product, which is blue in color and can be read spectrophotometrically. TMB has also been used in the quantitative determination of hemoglobin and in cytochemical staining for peroxidase. TMB is a hepatitis B antigen.

5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside is most commonly known as X-Gal. X-gal is a histochemical substrate for β-galactosidase which cleaves X-Gal to yield an insoluble blue precipitate.

X-Gal is a mainstay in molecular biology cloning applications in which it is used to detect the activity of β-galactosidase. X-Gal is used to detect the insertion of foreign DNA into the lacZ region of a plasmid DNA. Insertion of DNA into the lacZ region results in the loss of β-galactosidase activity. Bacteria cells that retain active β-galactosidase will result in characteristic blue colonies. Successful disruption of the lacZα gene disrupts the α-complementation of the β-galactosidase gene and the precipitate does not form, resulting in white colonies.

Many other applications also use X-Gal as a substrate to detect β-galactosidase activity. These include β-galactosidase -antibody linked immunoassays and immunohistochemistry, coliphage detection based on β-galactosidase induction and the detection of micrometastasis formation during tumor progession.

X-Gluc is a chromogenic substrate for beta-glucuronidase (GUS) which is encoded by the gusA (uid A) gene, a widely used reporter gene. Glucuronidase cleaves X-Gluc to produce colorless glucuronic acid and an intense blue precipitate. X-Gluc is often used in GUS staining of plant cells and tissue and in infections caused by E. coli.

X-pal is an off white to light brownish powder that is used as a chromogenic substrate for carboxylesterase. X-pal yields a blue precipitate when cleaved.

X-Sulfate K is a slightly off-white to faint-yellowish powder that is used as a chromogenic substrate for aryl-sulfatase. X-Sulfate K yields a blue precipitate when cleaved.

X-α-Gal is a chromogenic substrate for α-galactosidase, yielding a blue precipitate. X-α-Gal is used for detecting two-hybrid interactions directly on the culture media. This substrate eliminates the need for β-galactosidase liquid and filter-lift assays in two-hybrid analysis.
Used for species differentiation within the family Enterobacteriaceae and differentiation of Bifidobacteria species from Lactobacilli species.

α-L-Fucosidases are carbohydrate-processing enzymes belonging to the exoglycosidase family of glycoside hydrolases. These enzymes break down glycosidic bonds of sugars at the terminal ends. Fucose –containing glycolipids and glycoproteins can accumulate in different tissues when α-L-Fucosidase is inactive.

X-α-L-fucopyranoside is used as a chromogenic substrate for α-L-Fucosidase, producing a blue precipitate.

Y-β-D-GLC is a chromogenic substrate for β-D-glucosidase, yielding a blue precipitate upon cleavage.

α-D-Fucose is a six-carbon deoxy monosaccharide. It is equivalent to 6-deoxy-D-galactose. D-fucose acts as a non-metabolizable analogue of L-arabinose and inhibits induction of the L-arabinose operon in some bacteria. Some resistant bacteria possess a mutant araC gene where D-fucose acts as a gratuitous inducer of the L-arabinose operon. D-fucose induces β-galactosidase to some extent in Lactobacillus plantarum.

α-D-fucose inhibits the eukaryotic enzyme Lysine-specific demethylase 1 (LSD1A), thereby causing gene repression. α-D-fucose is being explored in treatments for neurodegenerative diseases because of its effect on gene repression. Seen upregulated in many cancers, LSD1A overexpression pushes the cell cycle of otherwise non-transformed cells so α-D-fucose is being explored in cancers treatments as well.